Advances in the Study of Schistosomiasis: the Postgenomic Era

Journal of Parasitology Research

Advances in the Study of Schistosomiasis: The Postgenomic Era

Guest Editors: Cristina Toscano Fonseca, Sergio Costa Oliveira, Andrea Teixeira-Carvalho, and Rashika El Ridi Advances in the Study of Schistosomiasis: The Postgenomic Era Journal of Parasitology Research

Advances in the Study of Schistosomiasis: The Postgenomic Era

This is a special issue published in “Journal of Parasitology Research.” All articles are open access articles distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Editorial Board

Takeshi Agatsuma, Japan Ana Maria Jansen, Brazil A. F. Petavy, France Jeﬀrey Bethony, USA MariaV.Johansen,Denmark Benjamin M. Rosenthal, USA Dave Chadee, USA Nirbhay Kumar, USA Joseph Schrevel, France Kwang Poo Chang, USA D. S. Lindsay, USA Jose´ F. Silveira, Brazil Wej Choochote, Thailand Bernard Marchand, France M. J. Stear, UK Alvin A. Gajadhar, Canada Renato A. Mortara, Brazil Xin-zhuan Su, USA C. Genchi, Italy Domenico Otranto, Italy Kazuyuki Tanabe, Japan Boyko B. Georgiev, Bulgaria Barbara Papadopoulou, Canada Contents

Advances in the Study of Schistosomiasis: The Postgenomic Era, Cristina Toscano Fonseca, Sergio Costa Oliveira, Andrea Teixeira-Carvalho, and Rashika El Ridi Volume 2013, Article ID 849103, 2 pages

Cytokine Pattern of T Lymphocytes in Acute Schistosomiasis mansoni Patients following Treated Praziquantel Therapy, Denise Silveira-Lemos, Matheus Fernandes Costa-Silva, Amanda Cardoso de Oliveira Silveira, Mauricio Azevedo Batista, Lucia´ Alves Oliveira-Fraga, Alda Maria Soares Silveira, Maria Carolina Barbosa Alvarez, Olindo Assis Martins-Filho, Giovanni Gazzinelli, Rodrigo Correa-Oliveira,ˆ and Andrea´ Teixeira-Carvalho Volume 2013, Article ID 909134, 13 pages

Cytokine and Chemokine Proﬁle in Individuals with Diﬀerent Degrees of Periportal Fibrosis due to Schistosoma mansoni Infection, Robson Da Paixao˜ De Souza, Luciana Santos Cardoso, Giuseppe Tittoni Varela Lopes, Maria Cec´ılia F. Almeida, Ricardo Riccio Oliveira, Leda Maria Alcantara,ˆ Edgar M. Carvalho, and Maria Ilma Araujo Volume 2012, Article ID 394981, 10 pages

New Frontiers in Schistosoma Genomics and Transcriptomics, Laila A. Nahum, Marina M. Mourao,˜ and Guilherme Oliveira Volume 2012, Article ID 849132, 11 pages

Changes in T-Cell and Monocyte Phenotypes In Vitro by Schistosoma mansoni Antigens in Cutaneous Leishmaniasis Patients, Aline Michelle Barbosa Baﬁca, Luciana Santos Cardoso, Sergio´ Costa Oliveira, Alex Loukas, Alfredo Goes,´ Ricardo Riccio Oliveira, Edgar M. Carvalho, and Maria Ilma Araujo Volume 2012, Article ID 520308, 10 pages

Transcriptional Profile and Structural Conservation of SUMO-Specific Proteases in Schistosoma mansoni, Roberta Verciano Pereira, Fernanda Janku Cabral, Matheus de Souza Gomes, Liana Konovaloff Jannotti-Passos, William Castro-Borges, and Renata Guerra-Sa´ Volume 2012, Article ID 480824, 7 pages

Schistosoma Tegument Proteins in Vaccine and Diagnosis Development: An Update, Cristina Toscano Fonseca, Gardeniaˆ Braz Figueiredo Carvalho, Clarice Carvalho Alves, and Tatiane Teixeira de Melo Volume 2012, Article ID 541268, 8 pages

Pathogenicity of Trichobilharzia spp. for Vertebrates,LichtenbergovaLucieandHor´ ak´ Petr Volume 2012, Article ID 761968, 9 pages Hindawi Publishing Corporation Journal of Parasitology Research Volume 2013, Article ID 849103, 2 pages http://dx.doi.org/10.1155/2013/849103

Editorial Advances in the Study of Schistosomiasis: The Postgenomic Era

Cristina Toscano Fonseca,1 Sergio Costa Oliveira,2 Andrea Teixeira-Carvalho,1 and Rashika El Ridi3

1 Centro de Pesquisas ReneRachou,Fundac´ ¸ao˜ Oswaldo Cruz, 30190-002 Belo Horizonte, MG, Brazil 2 Department of Biochemistry and Immunology, Federal University of Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil 3 Zoology Department, Faculty of Science, Cairo University, Giza 12613, Egypt

Correspondence should be addressed to Cristina Toscano Fonseca; [email protected]

Received 3 March 2013; Accepted 3 March 2013

Copyright © 2013 Cristina Toscano Fonseca et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Schistosomiasis remains a serious public health problem in In this special issue, a review by C. T. Fonseca et al. 76 countries and territories of the world, with more than givesanupdateonhowparasitegenomestogetherwith 200 million individuals infected, causing annual losses of 10.4 bioinformatics tools have helped in vaccine and diagnosis million in DALYs (disability adjusted to life years). Although development in the last few years. Since the parasite surface effective drugs against schistosomiasis are used in disease represents an interesting target for the host immune system, control programs, the prevalence of the disease remains this review was focused on parasite tegument as a source of unaltered, and new diseases foci and acute outbreaks are antigens. observed worldwide. Also, drug resistance by the parasites is Although the genome from the three most socioeconom- a concern, and new drugs and alternative control measures ically important schistosome species had already been pub- need to be developed. lished, many questions continue to challenge researchers. The An effective vaccine against schistosomiasis is an alterna- review by L. A. Nahum and coworkers discusses the advances tive and desirable tool to help in the disease control; however, on schistosome genomics and transcriptomics studies and thecomplexschistosomelifecycleandthecomplexityof point out the gaps and future directions in this research field. its interaction with the host immune system turn vaccine An example on how parasite genome database helps development into a difficult task. Neither drug development in understanding the parasite biology is presented in the nor vaccine development is sufficient to ensure the success paper by R. V. Pereira and colleagues. In their article, of schistosomiasis control programs. As a matter of fact, the using in silico analysis, they identified in the S. mansoni combination of an effective vaccine together with chemother- genome orthologs of the mammalian Sentrin/SUMO-specific apy is the strategy of choice. Many studies also demonstrate proteases (SENPs) 1 and 7,which are involved in many cellular that without a more sensitive diagnosis test, able to detect processes. The S. mansoni SENPs transcriptional profile as individuals with low parasite burden, no control strategy well as its structural conservation was analyzed. will achieve the desirable result which nowadays is diseases Just as important as understanding the biology of the elimination. parasite is deciphering how the host immune system deals The access to the genome from Schistosoma man- with schistosome infection and its relation to the pathogene- soni, Schistosoma japonicum, and Schistosoma haematobium sis of the disease. Clinically schistosomiasis is characterized recently published is expected to increase rapidly our under- bytwodistinctphases:theacuteandthechronicphase. standing of the parasite biology and also accelerate the The acute phase is commonly observed in individuals living development of new drugs, vaccine, and diagnosis methods in nonendemic area, while chronic phase is observed in by helping in the identification of new targets. endemic-area residents. 2 Journal of Parasitology Research

D. Silveira-Lemos et al. evaluated in their article immunological parameters in patients with acute schistosomiasis before and after praziquantel treatment. Interesting differences are demonstrated by the authors between noninfected, acute, and treated group, and a role for CD8+ T cells as a source of cytokines in acute patients is pointed by this study. The cytokine and chemokine profile observed in PBMCs from individuals infected with S. mansoni in response to specific stimulation and its correlation with different degrees of periportal fibrosis is described by R. P. de Souza and colleagues in their research article. Their results indicate potential biomarkers for the progression of liver pathology duetoschistosomiasis. Many schistosome antigens have been described to modulate inflammatory diseases such as asthma. A. M. B. Baficaetal.evaluatedintheirarticletheimpactofthe S. mansoni antigens: Sm29, TSP-2, and PIII in PBMCs culture from cutaneous leishmaniasis patients stimulated with L. braziliensis antigen. Their results demonstrated that schistosomeantigensinduceamodulatoryphenotypewith decreased monocyte activation and increased expression of T-lymphocyte modulatory molecules. Trichobilharzia spp. infection results in bird schistoso- miases, which causes severe pathogenic impact in birds and frequently results in cercarial dermatitis in humans. In their article, L. Lucie and H. Petr review the pathogenesis of bird schistosomiasis and the immune response triggered by Trichobilharzia spp. infection with emphasis on the new species T. regent. We hope you enjoy reading this special issue! Cristina Toscano Fonseca Sergio Costa Oliveira Andrea Teixeira-Carvalho Rashika El Ridi Hindawi Publishing Corporation Journal of Parasitology Research Volume 2013, Article ID 909134, 13 pages http://dx.doi.org/10.1155/2013/909134

Research Article Cytokine Pattern of T Lymphocytes in Acute Schistosomiasis mansoni Patients following Treated Praziquantel Therapy

Denise Silveira-Lemos,1, 2, 3, 4 Matheus Fernandes Costa-Silva,1, 2 Amanda Cardoso de Oliveira Silveira,1 Mauricio Azevedo Batista,4 Lúcia Alves Oliveira-Fraga,5 Alda Maria Soares Silveira,5 Maria Carolina Barbosa Alvarez,6 Olindo Assis Martins-Filho,1 Giovanni Gazzinelli,2 Rodrigo Corrêa-Oliveira,2 and Andréa Teixeira-Carvalho1

1 LaboratóriodeBiomarcadoresdeDiagnósticoeMonitoração,CentrodePesquisasRenéRachou,FIOCRUZ,BarroPreto, 30190-002 Belo Horizonte, MG, Brazil 2 Laboratório de Imunologia Celular e Molecular, Centro de Pesquisas René Rachou, FIOCRUZ, 30190-002 Belo Horizonte, MG, Brazil 3 Laboratório de Imunologia e Genômica de Parasitos, Departamento de Parasitologia, Instituto de Ciências Biológicas (ICB), Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil 4 Laboratório de Imunoparasitologia, Departamento de Ciências Biológicas, NUPEB, Instituto de Ciências Exatas e Biológicas, UniversidadeFederaldeOuroPreto,OuroPreto,MG,Brazil 5 Núcleo de Pesquisa em Imunologia, Faculdade de Ciências da Saúde, Universidade Vale do Rio Doce, Governador Valadares, MG, Brazil 6 Santa Casa de Misericórdia de Belo Horizonte, Belo Horizonte, MG, Brazil

Correspondence should be addressed to Denise Silveira-Lemos; [email protected]

Received 12 July 2012; Revised 24 September 2012; Accepted 26 September 2012

Academic Editor: Rashika El Ridi

Copyright © 2013 Denise Silveira-Lemos et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Acute schistosomiasis is associated with a primary exposure and is more commonly seen in nonimmune individuals traveling through endemic regions. In this study, we have focused on the cytokine pro�le of T lymphocytes evaluated in circulating leukocytes of acute Schistosomiasis mansoni-infected patients (ACT group) before and aer praziquantel treatment (ACT-TR group). Our data demonstrated increased values of total leukocytes, eosinophils, and monocytes in both groups. Interestingly, we have observed that patients treated with praziquantel showed increased values of lymphocytes as compared with noninfected group (NI) or ACT groups. Furthermore, a decrease of neutrophils in ACT-TR was observed when compared to ACT group. Analyses of short-term in vitro whole blood stimulation demonstrated that, regardless of the presence of soluble Schistosoma mansoni eggs antigen (SEA), increased synthesis of IFN- and IL-4 by T-cells was observed in the ACT group. Analyses of cytokine pro�le in CD� T cells demonstrated higher percentage of IFN- and IL-4 cells in both ACT and ACT-TR groups apart from increased percentage of IL- 10 cells only in the ACT group.𝛾𝛾 is study is the �rst one to point out the relevance of CD� T lymphocytes in the immune response induced during the acute phase of schistosomiasis.𝛾𝛾

1. Introduction the number of deaths caused by schistosomiasis is estimated at around 41.000 annually [1]. Schistosoma mansoni worms Schistosomiasis is a tropical parasitic chronic disease, caused dwell in peri-intestinal venules and cause intestinal, hepa- bywormsofthegenusSchistosoma. e infection is endemic tointestinal, and hepatosplenic schistosomiasis [2]. in tropics and subtropics around the world and is present Brazil is the country most affected by schistosomiasis in Africa, South America, Arabia, and Asia. ere are in the Americas, with 1.5 million people infected and more approximately 200 million people infected worldwide, and than 36 million at risk of acquiring infection [3, 4]. In the 2 Journal of Parasitology Research state of Minas Gerais, Schistosomiasis mansoni is prevalent in 2. Population, Material, and Methods 519 municipalities, with an estimated number of one million people infected [5]. 2.1. Study Population. e patients evaluated in this study e propagation and maintenance of schistosomiasis were infected accidentally by S. mansoni in the country in a given region are conditional of many factors such as village of São Geraldo da Piedade, an endemic area for schistosomiasis, located in the east of the state of Minas appropriate climate, socioeconomic conditions, rapid spread Gerais (MG), Brazil, during the traveling in a holiday. is of intermediate hosts, sanitary conditions, and water supply people reported swimming in a stream that probably harbors [6]. Tourist activity in endemic areas may be an important cercariae released by infected snails. In this area 60 of contributing factor to the propagation of outbreaks/cases of Biomphalaria glabrata are infected and the chronic disease schistosomiasis [7–10]. is frequent in the population. ese individuals went to% the In endemic areas for schistosomiasis, people develop the hospital because they felt very bad, with acute symptoms. e chronic phase of infection (which begins around six months patients did not live in areas of active transmission of S. man- aer exposure) and have different clinical manifestations. soni. All patients showed clinical symptoms associated with People who had never had previous contact with the parasite early S. mansoni infection such as fever, diarrhoea, headache, develop acute schistosomiasis when contracting the infection and nausea. Only patients who had positive results for quan- [11, 12]. titative parasitological stool examinations for S. mansoni by e acute phase starts aer cercarial infection, and the Kato-Katz method [20] and signed an informed consent local urticaria may appear in a few hours. Between one were included in this study. e group of acutely S. man- and four weeks aer infection, the migrating and maturing soni-infected patients (ACT) without praziquantel treatment schistosomula can cause a systemic hypersensitivity reaction consisted of twenty-one individuals, �ve women and sixteen with fever, general weakness, headache, nausea, vomiting, men, with ages ranging from 13 to 44 years, with parasite load diarrhea, and dry cough [2, 9]. In this phase, individuals ranging from 12 to 1812 eggs per gram of feces (epg). Aer present a mixed cytokine pro�le with predominance of 1 blood sample collection, all S. mansoni-infected individuals type of CD4 T-cell differentiation [13, 14]. received speci�c therapeutic treatment with praziquantel in the standard Brazilian dose (50–60 mg/Kg praziquantel), In the context+ of cytokine milieu triggered by S. man- regardless of whether or not they were going to participate soni infection, it has been suggested that interleukin (IL)-4 in this study [21]. One month aer praziquantel treatment, upregulates �broblast chemokine, matrix protein expression, blood was collected from a group of patients (ACT-TR) com- and collagen, which implies that IL-4 is a crucial cytokine posed of three women and four men, aged between 12 and for granuloma formation [15] and reduces the cellular 40 years. An additional noninfected group (NI) composed proliferative response to soluble egg antigens (SEA) [13]. of nineteen individuals, eleven woman and eight men, with IL-5 in schistosomiasis induces liver �brosis [16]. IL-10 age ranging from 19 to 45 years, who were healthy blood has been shown to be a major cytokine during infection donors contacted at the Hemominas Blood Bank Foundation with downregulatory activity of both 1 and 2 T cell in Belo Horizonte, MG, Brazil, participated in the study. All subpopulations [17]. Interferon gamma (IFN- ) is related to noninfected volunteers were included aer three consecutive the activation of macrophages and plays a key role in the negative parasitological exams for S. mansoni infection apart protective mechanism against periportal �brosis,𝛾𝛾 whereas the from negative serology for Chagas disease, leishmaniasis, proin�ammatory tumor necrosis factor-alpha (TNF- ) may human immunode�ciency virus infection, and hepatitis. is aggravate the disease [18]. study was approved by the Ethics Committees at the Oswaldo As regards the cytokine pro�le aer speci�c chemother-𝛼𝛼 Cruz Foundation (FIOCRUZ), the School of Medicine at the apy, it has been suggested that peripheral blood mononuclear Federal University of Minas Gerais (UFMG), and the Brazil- cells (PBMC) from patients with acute infection responded ian National Committee on Ethics in Research (CONEP). to SEA and soluble worm antigen preparation (SWAP) by producing signi�cantly higher amounts of IFN- and IL- 2.2. Ultrasonographic Analysis. Ultrasonographic evaluation 10. However, IL-5 was detected only in SEA-stimulated was performed in all individuals using a Nemio SSA/550A cultures, and little or no IL-4 was detected in SEA𝛾𝛾 or SWAP- ultrasound machine (Toshiba) with a 3 MHz sector probe. stimulated cells [19]. De Jesus et al. [9] suggested that Liver size, portal vein diameter, thickness of central walls most patients aer speci�c chemotherapy for schistosomiasis and peripheral portal branches, spleen size, and splenic vein spontaneously released high levels of TNF- , IL-1, and IL- diameter were assessed as described in previous studies [22, 6. In addition, detectable levels of IFN- were present in 23]. Liver span was measured both in the midclavicular the supernatants of unstimulated PBMC from𝛼𝛼 these patients. line and the midline. e liver was also examined for Stimulation of PBMC from patients with𝛾𝛾 acute disease only surface smoothness. Portal vein diameter was measured at its induced higher levels of IFN- upon SEA stimulation [9]. entrance into the liver and its bifurcation to the liver. Oblique In the present study, we evaluated the cytokine pro�le and longitudinal scans of the le upper quadrant were used (IFN- , TNF- , IL-4, IL-5,𝛾𝛾 and IL-10) of T lymphocytes to evaluate the spleen. e gallbladder was examined for and their subsets as well as the ultrasonographic features of wall thickness and stones. Periportal thickness was evaluated patients𝛾𝛾 with acute𝛼𝛼 schistosomiasis infection before and aer according to established criteria [22–24]. All individuals were praziquantel treatment. examined by the same physician. Journal of Parasitology Research 3

2.3. Evaluation of Hematological Parameters. Hemograms for intracellular cytokine, all cultures, including control and were performed with an automated blood cell counter (Coul- SEA stimulated, were treated with 2 mM EDTA (Sigma ter MD18, USA), using whole blood collected in 5 mL vacu- Chemical Company, St. Louis, MO, USA) and kept at room tainer tubes containing the anticoagulant ethylenediamine temperature for 15 min, to stop cell activation process. tetraacetic acid (EDTA) (Becton Dickinson Biosciences, San Following the short-term in vitro stimulation, cultured Diego, CA, USA). e parameters measured were total leuko- whole blood samples were washed with 6 mL of FACS buffer cytes counts and differential analysis of leukocyte subsets containing PBS supplemented with 0.5 Bovine Serum including the absolute counts of eosinophils, neutrophils, Albumin (BSA) and 0.1 sodium azide (Sigma Chemical lymphocytes, and monocytes. Company, St. Louis, MO, USA), by centrifugation% at 600 g at room temperature for% 7 min. Aer resuspension in 1 mL 2.4. Preparation of Antigens. S. mansoni eggs were isolated of FACS buffer, 400 L aliquots were dispensed into one 12× from the livers of infected mice, exposed 8 weeks previously 75 mm polystyrene tube (Becton Dickinson, Mountain View, to cercariae, homogenized, and ground in cold phosphate- CA, USA) and labeled𝜇𝜇 in the dark for 30 min at room temper-× buffered saline (PBS). e clear supernatant �uid result- ature with the manufacture’s recommended amount of mAbs ing from high-speed centrifugation of the homogenate at anti-CD3, anti-CD4, and anti-CD8-FITC. Following the 50.000 g for 1 h at room temperature was named soluble egg erythrocyte lyses step, the cells were incubated with 3 mL of antigens (SEA) and stored at 70 Cuntiluse[25]. FACS permeabilizing solution, containing FACS buffer supplemented with 0.5 of saponin (Sigma Chemical Company, × ∘ St. Louis, MO, USA) in the dark for 10 min at room temper- 2.5. Monoclonal Antibodies. −e experiments used mono- ature. Following incubation,% the samples were centrifuged at clonal antibodies (mAbs) against both human cell surface 600 g at room temperature for 7 min, the supernatant gently markers, including CD3 (UCTH-1), CD4 (SK3), and CD8 decanted and 3 mL of FACS buffer added to the resuspended (SK1) labeled with �uorescein isothiocyanate (FITC), and pellet.× Aer centrifugation, the pellet was resuspended and intracytoplasmic cytokines including IL-4 (MP4-25D2), IL- 30 L aliquots of cell suspension distributed in 96 wells U bot- 5 (TRFK5), IL-10 (JES3-9D7), IFN- (B27), and TNF- tom microplate (Falcon-Becton Dickinson, Mountain View, (Mab11) labeled with phycoerythrin (PE). Isotypic controls CA,𝜇𝜇 USA) and stained with 20 L of PE-labeled anticytokine were also used in all experiments, including𝛾𝛾 mouse IgG1𝛼𝛼 mAb (anti-IL-4, anti-IL-5, anti-IL-10, anti-IFN- , and anti- (679.1Mc7) and IgG2a (UCTH-1) labeled with FITC and PE, TNF- ,). e cells were incubated𝜇𝜇 in the dark for 30 min at respectively. All mAbs and controls were purchased from room temperature. Aer incubation, the cells were𝛾𝛾 washed Becton Dickinson Biosciences Pharmingen (San Diego, CA, with 150𝛼𝛼 L of FACS permeabilizing solution followed by USA). 200 L of FACS buffer. Aer washing, the stained cells were �xed in 200𝜇𝜇 L of FACS �x solution and the samples stored at 2.6. Short-Term Whole Blood Culture In Vitro and Intracel- 4 C𝜇𝜇 in the dark and analyzed by �ow cytometry. lular Cytokine Immunostaining. Short-term cultures in vitro ∘ 𝜇𝜇 and cytokine immunostaining were performed as described 2.7. Data Collection and Analysis. Immunostained samples by Silveira-Lemos et al. [26]. Brie�y, �ve hundred micro- were run in a FACScan three-color detection �ow cytometer liters of peripheral blood samples collected into Vacutainer (Becton Dickinson, San Jose, CA, USA). Data were collected tubes containing heparin sodium salt (Becton Dickinson, and analyzed with CellQuest soware (aer collection of Mountain View, CA, USA) were dispensed into individual 50.000 events/sample). Cytokine-expressing T lymphocytes, 17 100 mm polypropylene tubes (Falcon 2059 - Becton including CD3 , CD4 , and CD8 T cells, were identi�ed Dickinson, Mountain View, CA, USA). Short-term cultures by dual color immunophenotyping+ + + with FL1/FITC-labeled were× performed in the absence (control cultures) or in the antihuman cell surface markers against CD3, CD4 and presence of S. mansoni eggs derived antigens (SEA). For this CD8 mAbs together with FL2/PE-labeled anticytokine mAbs. purposes, 500 L of whole blood samples were incubated Lymphocyte gating was initially based on lymphocyte selec- in the presence of 500 L of RPMI 1640 plus Brefeldin A, tion by forward scatter (FSC) versus side light scatter (SSC) BFA (Sigma Chemical𝜇𝜇 Company, St Louis, MO, USA) at a properties on dot plot distributions, where they are con�ned �nal concentration of 10𝜇𝜇 g/mL. e blood samples were into a region of low size and complexity. Further analysis incubated for 4 h at 37 Cina5 CO humidi�ed incubator. of lymphocytes subpopulations was based on their selective staining with FITC-labeled anti-CD3, anti-CD4, and anti- ese conditions were∘ chosen𝜇𝜇 considering that the detection of cytokines, particularly in the% absence2 of exogenous stimuli, CD8 mAbs. Cytokine-expressing cell subsets were quanti�ed may re�ect the events of cytokine production by blood using FL1 cell surface markers versus FL2 anti cytokine-PE leukocytes in vivo. Antigen-speci�c stimulation was per- dot plots by setting quadrants to segregate FL2 positive and formed by previous in vitro incubation of 500 L whole blood negative cells based on the negative control immunostaining. aliquots in the presence of SEA at a �nal concentration of e results are expressed as the percentage of lymphocyte 25 g/mL for 1 h at 37 Cina5 CO humidi�ed𝜇𝜇 incubator. subsets expressing a given cytokine. Following the addition∘ of BFA (Sigma Chemical Company, St𝜇𝜇 Louis, MO, USA) at a �nal% concentration2 of 10 g/mL, 2.8. Statistical Analysis. e statistical analysis was per- blood samples were incubated for an additional 4 h at 37 C formed with soware GraphPad PRISM 5.00 for Windows in a 5 CO humidi�ed incubator. Prior to immunostaining𝜇𝜇 ∘ (La Jolla, CA, USA). Considering the parametric nature of the

% 2 4 Journal of Parasitology Research

T 1: Ultrasonographic features of the study group.

Site of measurement Reference values CT ( ) ACT ( ) Longitudinal le lobe of liver 107.0 102.2 16.0 (91.9–112.4) 99.7 20.9 (89.9–109.6) Anteroposterior le lobe of liver 70.0 42.7 4.4𝑁𝑁 (39.6–45.9) 푁푁푁 53.7 7.6 𝑁𝑁(50.2–57.3) 푁푁푁 Longitudinal right lobe of liver 150.0 134.3 ±10.5 (127.3–141.4) 147.4 ±11.5∗ (142.0–152.8) Anteroposterior right lobe of liver 100.0 70.3 ±10.6 (63.2–77.5) 84.3 ± 9.6∗ (79.7–89.0) Longitudinal spleen 120.0 88.1± 7.0 (83.4–92.9) 116.9 ± 15.6∗ (109.2–124.7) Anteroposterior spleen 70.0 33.7± 3.9 (31.0–36.3) 46.6 ± 8.5∗ (42.6–50.6) Portal vein 12.0 9.9 ± 1.2 (9.2–10.7) 10.5± 1.30∗ (9.9–11.1) Hilar portal vein wall 3.0 1.5± 0.3 (1.3–1.7) 2.0± 0.1 (1.9–2.1) Splenic vein < 9.0 6.4± 1.3 (5.5–7.2) 6.5± 0.9∗ (6.1–6.9) Superior mesenteric vein <9.0 6.4 ± 0.8 (5.9–7.1) 6.2± 0.8 (5.8–6.6) < ± ± Values are represented in mm as mean standard deviation and 95 con�dence intervals. Represents a statistically signi�cant difference ( )amonggroupsCTandACT.< ± ± ∗ ± % 𝑃𝑃 � 푃푃푃푃 data, all results were analyzed by the ANOVAtest followed by aer in vitro short-term incubation of whole blood samples Tukey�s multiple comparison test. Signi�cance was de�ned at focusing on T lymphocytes and their subsets (CD4 or CD8 . T cells). + + Regardless of the addition of SEA, the data revealed 3.𝑃𝑃 � Results푃푃푃푃 increased synthesis for IFN- and IL-4 by T cells in ACT as compared with NI (Figures 2(a) and 2(c), resp.). Aer 3.1. Ultrasonographic Features of the Study Group. Table 1 SEA stimulation, we also observed𝛾𝛾 increased synthesis of IL- shows the most important ultrasonographic features from 4 in ACT-TR as compared with NI (Figure 2(c)). Moreover, ACT in comparison with control group. Our data demon- the analysis of the impact of SEA addition to the cultures strated that ACT had signi�cant increase in the measurement detected increased synthesis of TNF- in ACT (Figure 2(b)). (in mm) of the longitudinal right lobe of liver (ACT: No signi�cant differences were found according to the sex ; CT: ), anteroposterior le lobe of liver (ACT: categories. 𝛼𝛼 ; CT: ), the anteroposterior right1 lobe47.4 of ± liver11.5 (ACT:134.3±10.5; CT: ), size of longitudinal and 3.4. Cytokine Producing T CD4 Lymphocytes following In the53.7 anteroposterior ± 7.6 42.7 spleen ± 4.4 (ACT: ; CT: Vitro SEA Stimulation. e analysis of the results showed, and ACT: 84.3±9.6 ; CT:70.3±10.6 , resp.), dimension of + in the control culture, increased synthesis of IL-4 by CD4 Hilar portal vein wall (ACT: 116.9; ± CT:15.6 88.1, resp.)± 7.0 T lymphocytes in ACT and ACT-TR groups as compared as compared46. with6 ± 8 the.5 CT group.33.7 ± No 3.9 signi�cant differences + with NI (Figure 3(c)). Furthermore, aer SEA stimulation, were found according to the sex2.0 categories.± 0.1 1.5 ± 0.3 we observed increased synthesis of TNF- by CD4 T lymphocytes in ACT as compared with the control culture+ 3.2. Hematological Parameters during Acute Schistosomiasis (Figure 3(b)). mansoni Infection before and aer Treatment with Praziquan- 𝛼𝛼 tel. e results showed an increase in the values of total leukocytes from ACT and ACT-TR as compared with NI. 3.5. Cytokine Producing T CD8 Lymphocytes following In A differential analysis of leukocyte subsets provides a more Vitro SEA Stimulation. e analysis+ of the cytokine pro�le in detailed description of the speci�c differences across cell the CD8 T subset revealed a higher number of differences types. e data showed increased counts of eosinophils and than in the+ CD4 T cells. Regardless of SEA stimulation, monocytes in the ACT and ACT-TR groups as compared increased synthesis+ of IFN- and IL-4 by CD8 T lympho- with NI. Moreover, increased values were detected for lym- cytes was observed in ACT as compared with+ NI (Figures phocytes from ACT-TR as compared with NI and ACT. 4(a) and 4(c), resp.). Moreover,𝛾𝛾 in cultures stimulated with Interestingly, aer speci�c therapy, count of neutrophils in SEA, increased synthesis of IL-4 was detected in ACT-TR ACT-TR was decreased in comparison with NI (Figure 1). as well as increased synthesis of IL-10 in ACT as compared with NI (Figures 4(c) and 4(e), resp.). Aer SEA stimulation, 3.3. Cytokine Producing T Lymphocytes following In Vitro data analysis also showed increased synthesis of IFN- or SEA Stimulation. Aiming to evaluate the impact of SEA IL-5 by CD8 T lymphocytes as compared with the control stimulation in triggering proin�ammatory and regulatory culture (Figures+ 4(a) and 4(d), resp.). When the impact𝛾𝛾 of cytokines by T lymphocytes and the effect of the speci�c SEA addition in the cultures from ACT-TR patient was therapy for this pro�le, we have performed a detailed single investigated, increased synthesis of IFN- or IL-4 by CD8 cell �ow cytometric analysis to �uantify the fre�uency of T lymphocytes were observed as compared with the control+ cytokine cells for IFN- , TNF- , IL-4, IL-5, and IL-10 cultures (Figures 4(a) and 4(c), resp.). 𝛾𝛾 + 𝛾𝛾 𝛼𝛼 Journal of Parasitology Research 5

Total leukocytes Eosinophils 20000 14000

) < 0.0001 ) = 0.0201 ॕ 3 ॕ 3 7500 ॕ = 0.0002 ॕ = 0.0012 10000 1000 500 Number of leukocytes of Number subpopulations (mm subpopulations Number of leukocytes of Number subpopulations (mm subpopulations

0 0 NI ACT ACT-TR NI ACT ACT-TR (a) (b) Neutrophils Lymphocytes 7000 6000 ) 3 ) 3 ॕ = 0.0068 ॕ = 0.0112 ॕ = 0.0014 3500 3000 Number of leukocytes of Number subpopulations (mm subpopulations Number of leukocytes of Number subpopulations (mm subpopulations

0 0 NI ACT ACT-TR NI ACT ACT-TR

750 ॕ = 0.0029 Number of leukocytes of Number subpopulations (mm subpopulations

0 NI ACT ACT-TR (e)

F 1: Analysis of hematological pro�le from patients with acute Schistosomiasis mansoni before (ACT, faint grey square = 21) and aer praziquantel treatment (ACT-TR, grey square = 07) and noninfected individuals (NI, white square = 19). e results are shown in bar-plot format highlighting the mean counts of datasets/mm standard deviation. �tatistically signi�cant diﬀerences (connecting lines) observed between groups NI, ACT, and ACT-TR were considered3 at . ± 𝑃𝑃 푃 푃푃푃푃 4. Discussion study such as headache, fever, diarrhea, and weight loss were consistent with those found by others authors [7, 9, 10, 29, Acute schistosomiasis is a severe disease and the mechanism 32]. involved in its clinical manifestations is not completely �etween two and eight weeks aer a �rst contact with understood. In the present study, we evaluated patients natural water infested by Schistosoma cercariae, susceptible exposed to the same place of contaminated water, 40–60 days aer exposure to cercaria. Diﬀerent studies concerning this infected patients may present a syndrome comprising of a acute disease refer to groups of tourists, �shermen, or sailors period of 2 to 30 days of fever, diarrhea, toxemia and weak- originally from a nonendemic country who has visited a ness, weight loss, abdominal pain, cough, myalgia, arthralgia, tropical zone [27–29]. However, as schistosomiasis is a focally edema, urticaria, nausea/vomiting, and hepatosplenomegaly distributed disease [29–31], the acute form is also diagnosed [33]. us, the diagnosis poses a challenge to physicians in inhabitants from endemic countries who do not live in owing to the nonspeci�city symptoms as well as the lack of endemic areas. e main clinical �ndings presented in our positivity for S. mansoni eggs in feces from acute patients in 6 Journal of Parasitology Research

IFN-ফ TNF-঩ 2.6 2.6

ॕ = 0.0395

ॕ = 0.0005 ॕ = 0.0006 T lymphocytes (%) T lymphocytes 1.3 (%) T lymphocytes 1.3 + + total total + + Cytokines 0 Cytokines 0 NI ACT ACT-TR NI ACT ACT-TR NI ACT ACT-TR NI ACT ACT-TR

Control culture SEA stimulation Control culture SEA stimulation (a) (b) IL-4 IL-5 2.6 2.6

ॕ = 0.0119

ॕ = 0.0001 ॕ < 0.0001 T lymphocytes (%) T lymphocytes T lymphocytes (%) T lymphocytes 1.3 1.3 + + total total + + Cytokines Cytokines 0 0 NI ACT ACT-TR NI ACT ACT-TR NI ACT ACT-TR NI ACT ACT-TR

Control culture SEA stimulation Control culture SEA stimulation (c) (d) IL-10 2.6

T lymphocytes (%) T lymphocytes 1.3 + total +

Cytokines 0 NI ACT ACT-TR NI ACT ACT-TR

Control culture SEA stimulation

(e)

F 2: Cytokine pattern of circulating T lymphocytes from patients with acute Schistosomiasis mansoni before (ACT, faint grey square = 21) and aer praziquantel treatment (ACT-TR, grey square = 07) and noninfected individuals (NI, white square = 19), following short-term in vitro cultivation in the absence (control culture) or presence of SEA stimulation. e results are shown in bar-plot format highlighting the mean percentage of datasets standard deviation. Statistically signi�cant di�erences (connecting lines) observed between groups NI, ACT, and ACT-TR were considered at . ± 𝑃𝑃 푃 푃푃푃푃 Journal of Parasitology Research 7

IFN-ফ TNF-঩ 2.2 2.2

ॕ = 0.0252 T lymphocytes (%) T lymphocytes

T lymphocytes (%) T lymphocytes 1.1

1.1 + + CD4 CD4 + +

Cytokines 0 Cytokines 0 NI ACT ACT-TR NI ACT ACT-TR NI ACT ACT-TR NI ACT ACT-TR

Control culture SEA stimulation Control culture SEA stimulation (a) (b) IL-4 IL-5 2.2 2.2

ॕ = 0.0169 T lymphocytes (%) T lymphocytes 1.1 = 0.0002 (%) T lymphocytes 1.1 + ॕ + CD4 CD4 + + Cytokines 0 Cytokines 0 NI ACT ACT-TR NI ACT ACT-TR NI ACT ACT-TR NI ACT ACT-TR

Control culture SEA stimulation Control culture SEA stimulation (c) (d) IL-10 2.2

T lymphocytes (%) T lymphocytes 1.1 + CD4 +

Cytokines 0 NI ACT ACT-TR NI ACT ACT-TR

Control culture SEA stimulation

(e)

F 3: Cytokine pattern of circulating T CD4 -lymphocytes from patients with acute Schistosomiasis mansoni before (ACT, faint grey square = 21) and aer praziquantel treatment (ACT-TR,+ grey square = 07) and noninfected individuals (NI, white square = 19), following short-term in vitro cultivation in the absence (control culture) or presence of SEA stimulation. e results are shown in bar-plot format highlighting the mean percentage of datasets standard deviation. Statistically signi�cant di�erences (connecting lines) observed between groups NI, ACT, and ACT-TR were considered at . ± 𝑃𝑃 푃 푃푃푃푃 8 Journal of Parasitology Research

IFN-ফ TNF-঩ 2.2 2.2 ॕ = 0.0381

ॕ = 0.0258 T lymphocytes (%) T lymphocytes T lymphocytes (%) T lymphocytes 1.1 1.1 + + ॕ = 0.0112

< 0.0001 CD8 CD8 ॕ + + Cytokines Cytokines 0 0 NI ACT ACT-TR NI ACT ACT-TR NI ACT ACT-TR NI ACT ACT-TR

Control culture SEA stimulation Control culture SEA stimulation (a) (b) IL-4 IL-5 2.2 2.2

ॕ = 0.0008

ॕ = 0.0173 ॕ = 0.0299 T lymphocytes (%) T lymphocytes T lymphocytes (%) T lymphocytes 1.1

1.1 + + ॕ < 0.0001 CD8 CD8 ॕ < 0.0001 + + Cytokines Cytokines 0 0 NI ACT ACT-TR NI ACT ACT-TR NI ACT ACT-TR NI ACT ACT-TR

Control culture SEA stimulation Control culture SEA stimulation (c) (d) IL-10 2.2

T lymphocytes (%) T lymphocytes 1.1 + ॕ = 0.0156 CD8 +

Cytokines 0 NI ACT ACT-TR NI ACT ACT-TR

Control culture SEA stimulation (e)

F 4: Cytokine pattern of circulating T CD8 -lymphocytes from patients with acute Schistosomiasis mansoni before (ACT, faint grey square = 21) and aer praziquantel treatment (ACT-TR,+ grey square = 07) and noninfected individuals (NI, white square = 19), following short-term in vitro cultivation in the absence (control culture) or presence of SEA stimulation. e results are shown in bar-plot format highlighting the mean percentage of datasets standard deviation. Statistically signi�cant di�erences (connecting lines) observed between groups NI, ACT, and ACT-TR were considered at . ± 𝑃𝑃 푃 푃푃푃푃 Journal of Parasitology Research 9 the earlier stage of the infection. In this context, abdominal in comparison with the control culture. Furthermore, in the ultrasound imaging can be a complementary tool to assist the control cultures, the results showed increased percentage of diagnosis of S. mansoni infection and its clinical monitoring. IL-4 T CD4 lymphocytes in both ACT and ACT-TR group e ultrasonographic �ndings presented in this study were in comparison+ + with NI (Figure 3). consistent with those described by Barata et al. [34] and e 1-2 model of CD4 T-cell differentiation is Costa-Silva [32], which showed nonspeci�c increase in the a well-established paradigm for understanding+ the basis of size of liver and spleen (Table 1). Costa-Silva [32]showedthat protective versus pathogenic immune responses for a host the ACT group had increased measurement of longitudinal of important human pathogens [43]. 1 cells secrete IFN- le/right lobe of liver, size of longitudinal spleen as well as and IL-2 and promote cell-mediated immunity, while 2 dimension of Hilar portal vein wall, and incipient periportal cells secrete IL-4, IL-5, and IL-10, among others. In this echogenic thickening, namely, grade I �brosis. us, the data 𝛾𝛾context, an important concept that evolved from the study presented here suggest that alterations identi�ed by analyses of 1 and 2 cell responses is the fact that both responses of ultrasonographic features, although not speci�c, are com- cross regulate each other. us, IFN- downregulates 2 patible with the acute phase of Schistosomiasis mansoni and cell development, while IL-4 and IL-10 antagonize 1 cell provide a reliable complementary tool for the diagnosis and differentiation [44]. In humans with acute𝛾𝛾 schistosomiasis, clinical followup of the disease. higher levels of IFN- and lower levels of IL-5 are found in Analyses of hematological parameters showed that ACT supernatants from PBMC compared to patients with chronic and ACT-TR groups presented increased values for total disease [19]. However,𝛾𝛾 the results by de Jesus et al. [9] showed leukocytes, eosinophils, or monocytes. On the other hand, only high production of the proin�ammatory cytokines IL- increased values for lymphocytes in addition to decreased 1, IL-6, and TNF- in cultures of unstimulated PBMC from values for neutrophils were observed only in the ACT-TR patients with acute schistosomiasis. us, our results suggest group (Figure 1). ese results suggest the existence of a sys- a mixed cytokine𝛼𝛼 pro�le produced by CD4 T cells. is temic activation of various hematopoietic lineages, antigen- cytokine milieu might be preventing tissue damage.+ In addi- induced secretion of parasite eggs [35–37]. Smithers et al. tion, development of a severe in�ammatory disease during [38] demonstrated in experimental models that the cellular acute schistosomiasis has been documented in knockout in�ltrate observed in hepatic granulomas is predominantly mice for IL-4 and IL-10 genes [45, 46]. Interestingly, our formed by eosinophils and monocytes. Different studies results demonstrated predominant increased percentage of showed that acute schistosomiasis patients have increased IFN- and IL-4 T CD8 lymphocytes, following short- levels of circulating eosinophils [26, 32, 39]. Previous studies term incubation+ of+ whole blood+ with SEA in both ACT and have also demonstrated that eosinophilia occurs between the ACT-TR𝛾𝛾 groups and increase of IL-10 cells only in the 5th and 7th weeks aer exposure to the parasite and may ACT group (Figure 4). is study is the+ �rst one to point be induced by 2 cytokines, such as IL-4, IL-10, IL-13, IL- out the relevance of CD8 T lymphocytes in the immune 9, and especially IL-5 [40]. Increased count of circulating response of acute phase of+ schistosomiasis. CD8 T cells monocytes was also observed by different studies [26, 41]. have been implicated in several immunopathological+ events Moreover, Borojevic et al. [41] showed a delayed differen- during helminthic infection including schistosomiasis [47]. tiation of bone marrow neutrophil granulocytes and blood e presence of CD8 T cells was observed in both early monocytosis of S. mansoni-infected animals. ese changes and chronic murine granulomas+ with an increased ratio of are compatible with modi�cations in the differentiation of CD8 cells during the chronic phase of the disease [48]. bone marrow myeloid precursors, favoring the production of Studies+ developed by Pancré et al. [49] in murine model a monomacrophage cell lineage. Indeed, some previous stud- demonstrated that SEA was able to stimulate IFN- or IL- ies show that during acute Schistosomiasis mansoni infection 2 producing CD8 T cells, suggesting a type 1 response a higher percentage of neutrophil apoptosis is found, com- induced by SEA. In+ humans, Oliveira-Prado et al. [50]𝛾𝛾 showed pared to patients with chronic illness or the control subjects a smaller percent of CD4 and a higher percent of CD8 [42]. Also, aer the praziquantel treatment, we have a higher cells in peripheral blood from+ patients with chronic schis-+ exposure of cryptic antigens which could be contributing to tosomiasis. Moreover, our group has previously described increase lymphocyte rather than neutrophil counts. Another the putative role of CD8 T subsets in controlling morbidity important point to be addressed is that, aer treatment, we during human schistosomiasis,+ which was evidenced by the have lesser levels of in�ammatory cytokines such as IL-1-beta increased levels of activated HLA-DR CD8 T lymphocytes and TNF- , important inducers of neutrophil mobilization to in patients presenting intestinal clinical+ form+ (INT) of the the peripheral blood during the acute phase of the disease. disease and low levels of CD28 CD8 cells in hepatosplenic Although𝛼𝛼 our results do not show increased values patients [51–53]. In addition,+ Teixeira-Carvalho+ et al. [54] for lymphocytes in the ACT group, in contrast with data observed increased percentage of IL-4 , IL-5 , and IL-10 obtained by Costa-Silva [32], a special interest in this study CD8 lymphocytes following short-term+ SEA stimulation+ of+ was the analysis of cytokines producing T lymphocytes, whole+ blood samples in vitro in the INT group. Although considering the importance of the speci�c immune response the recruitment of CD8 T cells by exogenous antigens (such to solve the infection. Analyses of proin�ammatory cytokines as those derived from +S. mansoni) primarily seems to be (IFN- and TNF- ) and regulatory cytokines (IL-4, IL-5, unexpected, recent studies have demonstrated that CD8 and IL-10) demonstrated higher percentage of TNF- T T cells are prone to respond to extracellular antigens in+ CD4 𝛾𝛾lymphocytes𝛼𝛼 in the ACT group aer SEA stimulation+ infectious diseases [55, 56] via bystander activation triggered + 𝛼𝛼 10 Journal of Parasitology Research by persistent antigenic stimulation, cytokines milieu [52, In conclusion, the data showed that a mixed cytokine 57, 58], or interaction with antigen-presenting cells (APC) pro�le is present during the acute phase of schistosomiasis that acquire exogenous antigens by phagocytosis and present mansoni. One month aer treatment with praziquantel there them throughout MHC class I molecules [59–62]. Similarly, is a increase in the production of IL-4 and IL-10 cytokines data of Montenegro et al. [19]showedthatPBMCfrom following SEA stimulation in circulating lymphocytes from acute schistosomiasis patients responded to SEA and SWAP infected patients. is �nding is associated with a reduction by producing signi�cantly higher amounts of IFN- and IL- in their morbidity. 10. A study carried out by de Jesus et al. [9] demonstrated the occurrence of higher levels of IFN- production𝛾𝛾 in Acknowledgments patients with acute schistosomiasis than those who have chronic infection. However, fewer patients with𝛾𝛾 acute disease eauthorswouldliketothankthetechnicalstaffofthe produced IL-10 in response to SEA. ese authors also Laboratório de Imunologia Celular e Molecular, Fundação showed a positive correlation between IL-10 levels in SEA- Oswaldo Cruz and Núcleo de Pesquisa em Imunologia, UNI- stimulated PBMC and time aer water exposure. On the VALE,Brazil, for their invaluable assistance during this study. other hand, Falcão et al. [63] showed that hepatosplenic eauthorsalsowishtothanktheProgramforTechno- schistosomiasis patients produced high levels of IFN- and logical Development in Public Health—PDTIS—FIOCRUZ low levels of IL-10 as compared to INT, associating this latter for use of its facilities. is study was supported by cytokine with the establishment/maintenance of the severe𝛾𝛾 FIOCRUZ, Conselho Nacional de Desenvolvimento Cien- clinical forms. Interestingly, studies by Pedras-Vasconcelos t��co e Tecnológico (CNPq), the Fundação de Amparo � and Pearce [64] demonstrated that mice infected with schis- Pesquisa de Minas Gerais (FAPEMIG), the National Insti- tosomes present a regulatory pathway in which type 1 CD8 tutes of Health NIH-ICIDR/Grant AI45451-01, and the cells, under the control of IL-4, dampen immunopathologic+ UNDP/World Bank/WHO Special Program for Research type 2 responses. Corrêa-Oliveira et al. [13]showedthat and Training in Tropical Diseases. O. A. Martins-Filho, A. the blockage of IL-4 and IL-5 using anti-IL-4 and anti-IL- Teixeira-Carvalho and R. Corrêa-Oliveira are thankful to 5 antibodies signi�cantly reduced the PBMC proliferative CNPq for the fellowships received (PQ). ATC is thankful to response to SEA antigens in acute, chronic intestinal, and the US Food and Drug Administration for the fellowship hepatosplenic patients. granted. Another interesting data found was the high IL-10 producing cells in response to SEA in uninfected individuals. References Like SEA is a complex mix of proteins, glycoproteins, and polysaccharides, this composition inducing could be induc- [1] WHO, World Health Organization, Geneva, Switzerland, ing cross-reactivity sharing some epitopes with other antigens 2012, http://whqlibdoc.who.int/publications/2010/ that previously sensitized the uninfected individuals. 9789241564090_eng.pdf. Studies show that praziquantel is an effective drug in [2] B. Gryseels, “Schistosomiasis,” International Journal of Infec- controlling infection with S. mansoni, promoting a reduction tious Diseases, vol. 26, pp. 383–397, 2012. in egg excretion and regression of �brosis in mice and [3] P. Steinmann, J. Keiser, R. Bos, M. Tanner, and J. Utzinger, humans [65–70]. We observed that one month aer speci�c “Schistosomiasis and water resources development: systematic chemotherapy with praziquantel, the patients in this study review, meta-analysis, and estimates of people at risk,” Lancet showed similar changes in the number of eosinophils and Infectious Diseases, vol. 6, no. 7, pp. 411–425, 2006. monocytes, but had a speci�c decrease in the number of [4] J. R. Lambertucci, “Acute schistosomiasis mansoni: revisited both neutrophils and lymphocytes as compared with NI and reconsidered,” Memorias Do Instituto Oswaldo Cruz, vol. and ACT or only NI, respectively (Figure 1). Some authors 105, no. 4, pp. 422–435, 2010. have demonstrated that chemotherapy-induced immune [5] N. Katz, “Schistosomiasis control in Brazil,” Memorias Do responses are heterogeneous, depending on the type of Instituto Oswaldo Cruz, vol. 93, pp. 33–35, 1998. antigen used and time of analysis aer treatment [70, 71]. [6] R. S. Do Amaral, P. L. Tauil, D. D. Lima, and D. Engels, In this context, we observed a higher number of IFN- and “An analysis of the impact of the Schistosomiasis Control IL-4 T CD8 lymphocytes in response to SEA in the ACT- Programme in Brazil,” Memorias Do Instituto Oswaldo Cruz, + vol. 101, no. 1, pp. 79–85, 2006. TR group+ (Figure+ 4). De Souza et al. [72] demonstrated—six𝛾𝛾 months aer speci�c treatment with oxamniquine—that the [7] C. S. Barbosa, S. M. L. Montenegro, F. G. C. Abath, and A. L. Coutinho Domingues, “Speci�c situations related to acute IFN- levels increased signi�cantly in response to SEA. schistosomiasis in Pernambuco, Brazil,” Memorias Do Instituto Similarly, it was reported that patients with chronic disease Oswaldo Cruz, vol. 96, pp. 169–172, 2001. living in endemic areas showed increased levels of IFN- aer 𝛾𝛾 [8] C. S. Barbosa, A. L. Domingues, F. Abath et al., “An outbreak of treatment in response to parasite antigens [73]. Probably, acute schistosomiasis at Porto de Galinhas beach, Pernambuco, the increase in T-cell reactivity aer chemotherapy can𝛾𝛾 be Brazil,” Cadernos De Saude Publica, vol. 17, no. 3, pp. 725–728, explained by exposure of released antigens to the immune 2001. system following the destruction of worms by chemotherapy [9] A. R. De Jesus, A. Silva, L. B. Santana et al., “Clinical and [43, 73–76]. In contrast to our study, De Souza et al. [72] immunologic evaluation of 31 patients with acute schistosomi- have not found increased levels of IL-4 in patients with acute asis mansoni,” Journal of Infectious Diseases, vol. 185, no. 1, pp. schistosomiasis aer treatment with oxamniquine. 98–105, 2002. Journal of Parasitology Research 11

[10] M. J. Enk, R. L. Caldeira, O. S. Carvalho, and V.T. Schall, “Rural [25] G. Gazzinelli, N. Katz, R. S. Rocha, and D. G. Colley, “Immune tourism as risk factor for the transmission of schistosomiasis in responses during human schistosomiasis mansoni. X. Pro- Minas Gerais, Brazil,” Memorias Do Instituto Oswaldo Cruz, vol. duction and standardization of an antigen-induced mitogenic 99, no. 5, pp. 105–108, 2004. activity by peripheral blood mononuclear cells from treated, but [11] R. Veronsi and R. Focaccia, Tratado De Infectologia, Atheneu, not active cases of schistosomiasis,” Journal of Immunology, vol. Rio de Janeiro, Brazil, 2 edition, 2002. 130, no. 6, pp. 2891–2895, 1983. [12] L. Q. Nguyen, J. Estrella, E. A. Jett, E. L. Grunvald, L. Nichol- [26] D. Silveira-Lemos, A. Teixeira-Carvalho, A. O. Martins-Filho et son, and D. L. Levin, “Acute schistosomiasis in nonimmune al., “Eosinophil activation status, cytokines and liver �brosis in travelers: Chest CT �ndings in 10 patients,” American Journal Schistosoma mansoni infected patients,” Acta Tropica, vol. 108, of Roentgenology, vol. 186, no. 5, pp. 1300–1303, 2006. no. 2-3, pp. 150–159, 2008. [13] R. Corrêa-Oliveira, L. C. C. Malaquias, P. L. Falcão et al., [27] M. N. Lunde and E. A. Ottesen, “Enzyme-linked immunosor- “Cytokines as determinants of resistance and pathology in bent assay (ELISA) for detecting IgM and IgE antibodies in human Schistosoma mansoni infection,” Brazilian Journal of human schistosomiasis,” American Journal of Tropical Medicine Medical and Biological Research, vol. 31, no. 1, pp. 171–177, and Hygiene, vol. 29, no. 1, pp. 82–85, 1980. 1998. [28] B. Evengard, L. Hammarstrom, C. I. E. Smith, and E. Linder, [14] S. A. Rezende, J. R. Lambertucci, and A. M. Goes, “Role of “Early antibody responses in human schistosomiasis,” Clinical immune complexes from pacients with different clinical forms and Experimental Immunology, vol. 80, no. 1, pp. 69–76, 1990. of schistosomiasis in the modulation of in vitro granuloma [29] A. Rabello, “Acute human schistosomiasis mansoni,” Memórias reaction,” Memorias Do Instituto Oswaldo Cruz,vol.92,no.5, Do Instituto Oswaldo Cruz, vol. 90, no. 2, pp. 277–280, 1995. pp. 683–687, 1997. [30] S. B. Pessoa and J. P. Amorim, “Notas sobre a esquistosso- [15] X. Liu, T. Kohyama, H. Wang et al., “2 cytokine regulation mose mansônica em algumas localidades de Alagoas,” Revista of type I collagen gel contraction mediated by human lung Brasileira De Medicina, vol. 14, pp. 420–422, 1957. mesenchymal cells,” American Journal of Physiology, vol. 282, [31] K. Kloetzel, “Schistosomiasis in Brazil: does social development no. 5, pp. L1049–L1056, 2002. suffice?” Parasitology Today, vol. 5, no. 12, pp. 388–391, 1989. [16] R. M. Reiman, R. W. ompson, C. G. Feng et al., “Interleukin- [32] M. F. Costa-Silva, Acompanhamento clínico, epidemiológico e 5 (IL-5) augments the progression of liver �brosis by regulating imunológico de pacientes portadores da fase aguda da esquis- IL-13 activity,” Infection and Immunity, vol. 74, no. 3, pp. tossomose mansoni, su�metidos � terap�utica especí�ca com 1471–1479, 2006. Praziquantel [Dissertação (Mestrado em Doenças Infecciosas e [17] T. R. Mosmann and K. W. Moore, “e role of IL-10 in cross- Parasitárias)], Fundação Oswaldo Cruz. Centro de Pesquisas regulation of TH1 and TH2 responses,” Immunology Today, vol. René Rachou. Programa de Pós-graduação em Ciências da 12, no. 3, pp. A49–A53, 1991. Saúde, Belo Horizonte, Brazil, 2009, http://www.cpqrr.�o- [18] M. Booth, J. K. Mwatha, S. Joseph et al., “Periportal �brosis in cruz.br/texto-completo/D_5.pdf. human Schistosoma mansoni infection is associated with low [33] J. R. Coura and R. S. Amaral, “Epidemiological and control IL-10, Low IFN- , high TNF- , or low RANTES, depending aspects of schistosomiasis in Brazilian endemic areas,” Memo- on age and gender,” Journal of Immunology, vol. 172, no. 2, pp. rias Do Instituto Oswaldo Cruz, vol. 99, no. 5, pp. 13–19, 2004. 1295–1303, 2004.𝛾𝛾 𝛼𝛼 [34] C. H. Barata, R. A. Pinto-Silva, and J. R. Lambertucci, “Abdom- [19] S. M. L. Montenegro, P. Miranda, S. Mahanty et al., “Cytokine inal ultrasound in acute schistosomiasis mansoni,” British production in acute versus chronic human schistosomia- Journal of Radiology, vol. 72, pp. 949–952, 1999. sis mansoni: the cross-regulatory role of interferon- and [35] G. J. Race, R. M. Michaels, J. H. Martin, J. E. Larsh Jr., and J. L. interleukin-10 in the responses of peripheral blood mononu- Matthews, “Schistosoma mansoni eggs: an electron microscopic clear cells and splenocytes to parasite antigens,” Journal𝛾𝛾 of study of shell pores and microbarbs,” Proceedings of the Society Infectious Diseases, vol. 179, no. 6, pp. 1502–1514, 1999. for Experimental Biology and Medicine, vol. 130, no. 3, pp. [20] N. Katz, A. Chaves, and J. Pellegrino, “A simple device for 990–992, 1969. quantitative stool thick-smear technique in schistosomiasis [36] A. Sher, R. L. Coffman, S. Hieny, P. Scott, and A. W. Cheever, m an s on i ,” Revista Do Instituto de Medicina Tropical de Sao “Interleukin 5 is required for the blood and tissue eosinophilia Paulo, vol. 14, no. 6, pp. 397–400, 1972. but not granuloma formation induced by infection with Schisto- [21] J. G. Hardman, L. E. Limbird, and A. G. Gilman, Eds., Goodman soma mansoni,” Proceedings of the National Academy of Sciences & Gilman’s.e Pharmacological Basis of erapeutics, vol. 2045, of the United States of America, vol. 87, no. 1, pp. 61–65, 1990. McGraw-Hill, New York, NY, USA, 10th edition, 2001. [37] H. L. Lenzi, R. G. Pacheco, M. Pelajo-Machado, M. S. Panasco, [22] M. F. Abdel-Wahab, G. Esmat, A. Farrag, Y. A. El-Boraey, and W. S. Romanha, and J. A. Lenzi, “Immunological system and G. T. Strickland, “Grading of hepatic schistosomiasis by the use Schistosoma mansoni: co-evolutionary immunobiology. what is of ultrasonography,” American Journal of Tropical Medicine and the eosinophil role in parasite-host relationship?” Memorias Do Hygiene, vol. 46, no. 4, pp. 403–408, 1992. Instituto Oswaldo Cruz, vol. 92, pp. 19–32, 1997. [23] M. Homeida, A. F. Abdel-Gadir, A. W. Cheever et al., “Diag- [38] S. R. Smithers, D. J. McLaren, and F. J. Ramalho-Pinto, nosis of pathologically con�rmed Symmers� periportal �brosis “Immunity to schistosomes: the target,” e American Journal by ultrasonography: a prospective blinded study,” American of Tropical Medicine and Hygiene, vol. 26, no. 6, pp. 11–19, 1977. JournalofTropicalMedicineandHygiene, vol. 38, no. 1, pp. [39] E. Caumes and M. Vidailhet, “Acute neuroschistosomiasis: a 86–91, 1988. cerebral vasculitis to treat with corticosteroids not praziquan- [24] G. G. Cerri, V. A. Alves, and A. Magalhaes, “Hepatosplenic t e l ,” Journal of Travel Medicine, vol. 17, no. 5, pp. 359–361, 2010. schistosomiasis mansoni: ultrasound manifestations,” Radiol- [40] D. C. Cara, D. Negrao-Correa, and M. M. Teixeira, “Mech- ogy, vol. 153, no. 3, pp. 777–780, 1984. anisms underlying eosinophil trafficking and their relevance 12 Journal of Parasitology Research

i n v i v o,” Histology and Histopathology, vol. 15, no. 3, pp. CD4 CD25HIGH T-cells and clinical forms of human 899–920, 2000. schistosomiasis,”+ + Acta Tropica, vol. 108, no. 2-3, pp. 139–149, [41] R. Borojevic, C. G. Pinto, M. C. el-Cheikh, and H. S. Dutra, 2008. “Experimental murine schistosomiasis mansoni: hyperplasia of [55] J. M. M. Den Haan and M. J. Bevan, “Antigen presentation the mono-macrophage cell lineage and stimulation of myeloid to CD8 T cells: cross-priming in infectious diseases,” Current proliferation by peripheral macrophages,” Brazilian Journal of Opinion+ in Immunology, vol. 13, no. 4, pp. 437–441, 2001. Medical and Biological Research, vol. 22, no. 5, pp. 579–586, [56] B. J. Manfras, S. Reuter, T. Wendland, and P. Kern, “Increased 1989. activation and oligoclonality of peripheral CD8 T cells in the [42] I. M. el-Sharkawy, A. A. Ibrahim, and M. A. el-Bassuoni, “Neu- chronic human helminth infection alveolar echinococcosis,”+ trophil apoptosis in acute and chronic Schistosoma mansoni Infection and Immunity, vol. 70, no. 3, pp. 1168–1174, 2002. infection,” Journal of the Egyptian Society of Parasitology, vol. [57] A. Kalinkovich, Z. Weisman, Z. Greenberg et al., “Decreased 32, no. 2, pp. 507–516, 2002. CD4 and increased CD8 counts with T cell activation is [43] T. R. Mosmann, L. Li, H. Hengartner, D. Kagi, W. Fu, and associated with chronic helminth infection,” Clinical and Exper- S. Sad, “Differentiation and functions of T cell subsets,” CIBA imental Immunology, vol. 114, no. 3, pp. 414–421, 1998. Foundation Symposia, vol. 204, pp. 148–158, 1997. [58] G. Borkow, Q. Leng, Z. Weisman et al., “Chronic immune [44] T. R. Mosmann and S. Sad, “e expanding universe of T-cell activation associated with intestinal helminth infections results subsets: 1, 2 and more,” Immunology Today,vol.17,no.3, in impaired signal transduction and anergy,” Journal of Clinical pp. 138–146, 1996. Investigation, vol. 106, no. 8, pp. 1053–1060, 2000. [45] L. R. Brunet, F. D. Finkelman, A. W.Cheever, M. A. Kopf, and E. [59] E. P.Grant and K. L. Rock, “MHC class I-restricted presentation J. Pearce, “IL-4 protects against TNF- -mediated cachexia and of exogenous antigen by thymic antigen-presenting cells in vitro death during acute schistosomiasis,” Journal of Immunology, vol. and in vivo,” Journal of Immunology, vol. 148, no. 1, pp. 13–18, 1992. 159, no. 2, pp. 777–785, 1997. 𝛼𝛼 [60] K. L. Rock, S. Gamble, L. Rothstein, C. Gramm, and B. [46] P. G. Fallon, E. J. Richardson, P. Smith, and D. W. Dunne, Benacerraf, “Dissociation of -microglobulin leads to the “Elevated type 1, diminished type 2 cytokines and impaired accumulation of a substantial pool of inactive class I MHC antibody response are associated with hepatotoxicity and mor- heavy chains on the cell surface,” Cell2 , vol. 65, no. 4, pp. 611–620, talities during Schistosoma mansoni infection of CD4-depleted 𝛽𝛽 1991. mice,” European Journal of Immunology, vol. 30, no. 2, pp. 470–480, 2000. [61] K. L. Rock, C. Fleischacker, and S. Gamble, “Peptide-priming of cytolytic T cell immunity in vivo using - microglobulin as an [47] P.Raso, “Esquistossomose mansônica,”in Patologia, L. Bogliolo, adjuvant,” Journal of Immunology, vol. 150, no. 4, pp. 1244–1252, Ed., pp. 534–545, Guanabara Koogan, Rio de Janeiro, Brazil, 4th 1993. 2 edition, 1994. 𝛽𝛽 [62] M. Larsson, J. F. Fonteneau, and N. Bhardwaj, “Dendritic cells [48] P.J. Perrin and S. M. Phillips, “e molecular basis of granuloma resurrect antigens fromdead cells,” Trends in Immunology, vol. formation in schistosomiasis. I. A T cell-derived suppressor 22, no. 3, pp. 141–148, 2001. effector factor,” Journal of Immunology, vol. 141, no. 5, pp. [63] P. L. Falcão, L. C. Malaquias, O. A. Martins-Filho et al., 1714–1719, 1988. “Human schistosomiasis mansoni: IL-10 modulates the in vitro [49] V. Pancré, M. Delacre, J. Herno, and C. Auriault, “Schistosomal granuloma formation,” Parasite Immunology, vol. 20, no. 10, pp. egg antigen-responsive CD8 T-cell population in Schistosoma 447–454, 1998. mansoni-infected BALB/c mice,” Immunology, vol. 98, no. 4, pp. [64] J. A. Pedras-Vasconcelos and E. J. Pearce, “Type 1 CD8 T 525–534, 1999. Cell responses during infection with the helminth Schistosoma+ [50] R. Oliveira-Prado, I. R. Caldas, A. Teixeira-Carvalho et al., mansoni,” Journal of Immunology, vol. 157, no. 7, pp. 3046–3053, “CD4 and CD8 distribution pro�le in individuals infected 1996. by Schistosoma+ mansoni+ ,” Scandinavian Journal of Immunology, [65] E. Doehring-Schwerdtfeger, I. M. Abdel-Rahim, R. Kardorff et vol. 69, no. 6, pp. 521–528, 2009. al., “Ultrasonographical investigation of periportal �brosis in [51] O. A. Martins-Filho, J. R. Mello, and R. Correa-Oliveira, “e children with Schistosoma mansoni infection: reversibility of spleen is an important site of T cell activation during human morbidity twenty-three months aer treatment with praziquan- hepatosplenic schistosomiasis,” Memorias Do Instituto Oswaldo t e l ,” American Journal of Tropical Medicine and Hygiene, vol. 46, Cruz, vol. 93, supplement 1, pp. 159–164, 1998. no. 4, pp. 409–415, 1992. [52] O. A. Martins-Filho, J. R. Cunha-Melo, J. R. Lambertucci et [66] G. F. Cota, R. A. Pinto-Silva, C. M. Antunes, and J. R. Lamber- al., “Clinical forms of human Schistosoma mansoni infection tucci, “Ultrasound and clinical investigation of hepatosplenic are associated with differential activation of T-cell subsets and schistosomiasis: evaluation of splenomegaly and liver �brosis costimulatory molecules,” Digestive Diseases and Sciences, vol. four years aer mass chemotherapy with oxamniquine,” Ameri- 44, no. 3, pp. 570–577, 1999. can Journal of Tropical Medicine and Hygiene,vol.74,no.1,pp. [53] A. Teixeira-Carvalho, O. A. Martins-Filho, Z. A. Andrade, J. 103–107, 2006. R. Cunha-Mello, R. A. Wilson, and R. Corrêa-Oliveira, “e [67] Z. A. Andrade, A. P.Baptista, and T. S. Santana, “Remodeling of study of T-cell activation in peripheral blood and spleen of hepatic vascular changes aer speci�c chemotherapy of schisto- hepatosplenic patients suggests an exchange of cells between somal periportal �brosis,” Memorias Do Instituto Oswaldo Cruz, these two compartments in advanced human schistosomiasis vol. 101, no. 1, pp. 267–272, 2006. mansoni infection,” Scandinavian Journal of Immunology, vol. [68] J. H. Kihara, N. Muhoho, D. Njomo et al., “Drug efficacy 56, no. 3, pp. 315–322, 2002. of praziquantel and albendazole in school children in mwea [54] A. Teixeira-Carvalho, O. A. Martins-Filho, V. Peruhype- division, central province, kenya,” Acta Tropica, vol. 102, no. 3, Magalhães et al., “Cytokines, chemokine receptors, pp. 165–171, 2007. Journal of Parasitology Research 13

[69] P. Martins-Leite, G. Gazzinelli, L. F. Alves-Oliveira et al., “Eﬀect of chemotherapy with praziquantel on the production of cytokines and morbidity associated with schistosomiasis m an s on i ,” Antimicrobial Agents and Chemotherapy, vol. 52, no. 8, pp. 2780–2786, 2008. [70] C. M. Fitzsimmons, S. Joseph, F. M. Jones et al., “Chemotherapy for schistosomiasis in ugandan �shermen� treatment can cause a rapid increase in interleukin-5 levels in plasma but decreased levels of eosinophilia and worm-speci�c immunoglobulin E,” Infection and Immunity, vol. 72, no. 7, pp. 4023–4030, 2004. [71] S. Joseph, F. M. Jones, K. Walter et al., “Increases in human T helper 2 cytokine responses to Schistosoma mansoni worm and worm-tegument antigens are induced by treatment with praziquantel,” Journal of Infectious Diseases,vol.190,no.4,pp. 835–842, 2004. [72] J. R. De Souza, C. N. Morais, M. L. Aroucha et al., “Treatment of human acute schistosomiasis with oxamniquine induces an increase in interferon- response to Schistosoma mansoni antigens,” Memorias Do Instituto Oswaldo Cruz, vol. 102, no. 2, pp. 225–228, 2007. 𝛾𝛾 [73] R. Corrêa-Oliveira, I. Rodrigues Caldas, O. A. Martins-Filho et al., “Analysis of the eﬀects of treatment of human Schistosoma mansoni infection on the immune response of patients from endemic areas,” Acta Tropica, vol. 77, no. 1, pp. 141–146, 2000. [74] M. I. Araújo, A. R. De Jesus, O. Bacellar, E. Sabin, E. Pearce, and E. M. Carvalho, “Evidence of a T helper type 2 activation in human schistosomiasis,” European Journal of Immunology, vol. 26, no. 6, pp. 1399–1403, 1996. [75] J. L. Grogan, P. G. Kremsner, A. M. Deelder, and M. Yaz- danbakhsh, “Elevated proliferation and interleukin-4 release from CD4 cells aer chemotherapy in human schistosoma haematobium+ infection,” European Journal of Immunology, vol. 26, no. 6, pp. 1365–1370, 1996. [76] C. F. Brito, I. R. Caldas, P. Coura Filho, R. Corrêa-Oliveira, and S. C. Oliveira, “CD4 T cells of schistosomiasis natu- rally resistant individuals living+ in an endemic area produce interferon- and tumour necrosis factor- in response to the recombinant 14KDA Schistosoma mansoni fatty acid-binding protein,” Scandinavian𝛾𝛾 Journal of Immunology𝛼𝛼 , vol. 51, no. 6, pp. 595–601, 2000. Hindawi Publishing Corporation Journal of Parasitology Research Volume 2012, Article ID 394981, 10 pages doi:10.1155/2012/394981

Research Article Cytokine and Chemokine Proﬁle in Individuals with Different Degrees of Periportal Fibrosis due to Schistosoma mansoni Infection

Robson Da Paixao˜ De Souza,1 Luciana Santos Cardoso,1, 2, 3 Giuseppe Tittoni Varela Lopes,1 Maria Cecılia´ F. Almeida,1 Ricardo Riccio Oliveira,1, 3, 4 Leda Maria Alcantara,ˆ 1 Edgar M. Carvalho,1, 3, 4, 5 and Maria Ilma Araujo1, 3, 4, 5

1 Serviço de Imunologia, Complexo Hospitalar Universitario´ Professor Edgard Santos, Universidade Federal da Bahia, Rua Joao˜ das Botas s/n, Canela, 40.110-160 Salvador, BA, Brazil 2 Departamento de Ciências da Vida, Universidade do Estado da Bahia, 2555 Rua Silveira Martins, Cabula, 41.150-000 Salvador, BA, Brazil 3 Instituto Nacional de Ciência e Tecnologia em Doenças Tropicais (INCT-DT-CNPQ/MCT), Rua Joao˜ das Botas s/n, Canela, 40.110-160 Salvador, BA, Brazil 4 Departamento de Analises´ Cl´ınicas e Toxicologicas,´ Faculdade de Farmacia,´ UFBA, Avenida Adhemar de Barros s/n, Ondina, 40.170-970 Salvador, BA, Brazil 5 Escola Bahiana de Medicina e Saude´ Publica,´ Rua Frei Henrique No. 08, Nazaré, 40.050-420 Salvador, BA, Brazil

Correspondence should be addressed to Robson Da Paixao˜ De Souza, [email protected]

Received 13 July 2012; Accepted 7 November 2012

Academic Editor: Sergio Costa Oliveira

Copyright © 2012 Robson Da Paixao˜ De Souza et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Periportal fibrosis in schistosomiasis has been associated to the host immune response to parasite antigens. We evaluated the immune response in S. mansoni infected individuals with different degrees of periportal fibrosis. Cytokine and chemokines were measured in serum and in supernatants of PBMC cultures stimulated with the soluble adult worm (SWAP) or egg (SEA) antigens, using a sandwich ELISA. The levels of IL-5 in response to SEA were higher in individuals with moderate to severe fibrosis (310.9 pg/mL) compared to individuals without fibrosis (36.8 pg/mL; P = 0.0418). There was also a higher production of TNF-α in cultures stimulated with SWAP in patients with insipient fibrosis (1446 pg/mL) compared to those without fibrosis (756.1 pg/mL; P = 0.0319). The serum levels of IL-13 and MIP-1α were higher in subjects without fibrosis than in those with moderate to severe fibrosis. However a positive association between serum levels of IL-13, TNF-α, MIP-1α, and RANTES and S. mansoni parasite burden was found. From these data we conclude that IL-5 and TNF-α may participate in liver pathology in schistosomiasis. The positive association between IL-13, TNF-α, MIP-1α, and RANTES with parasite burden, however, might predict the development of liver pathology.

1. Introduction million are at risk of infection [3]. The liver pathology results from the host immune response to parasite antigens from the Schistosomiasis is a chronic parasitic infection that affects eggs that become trapped in the portal venous system, and 200 million people in Africa, South America, and Asia and it is associated to the morbidity and mortality described in it is estimated that roughly 700 million people in the world schistosomiasis [4]. live at risk of infection [1, 2]. In Brazil, the only species The granuloma formation around S. mansoni eggs is described is Schistosoma mansoni, and it is estimated that complex and represents an interaction between products seven million people are infected by the parasite and about 25 secreted by the miracidia, which are released from the egg 2 Journal of Parasitology Research and the host immune response [5]. Consequently, the gran- The inclusion criteria for this study were individuals from ulomas formed act as barriers that prevent the dispersion of endemic areas who have at least one positive parasitological egg antigens of S. mansoni, and the consequent damage to exam for S. mansoni. From the 537 individuals who agreed the liver parenchyma. However, when caused by deposition to participate in this study 334 were infected with S. mansoni of large numbers of eggs, the inflammatory process may (62.5%). The frequency of other helminthic infections was progress to severe fibrosis, which leads to interruption of 43.4% for Trichuris trichiura, 37.4% for Ascaris lumbricoides, normal blood flow in the venous system to the sinusoids 33.7% for Hookworms, and 3.5% for Strongyloides stercoralis. resulting in portal hypertension, hepatosplenomegaly, and From 334 individuals who were infected with S. mansoni, formation of gastric and esophageal varices that can lead 220 agreed to perform abdominal ultrasound, in order to to bleeding and even death. This severe form of the disease determine the degree of periportal fibrosis. They also agreed occurs in five to ten percent of infected subjects living in to donate blood for the study of the immunological response. endemic areas [6–8]. For this particular aim, we did not include individuals under Some studies have evaluated the immune response five or above 60 years old. We also did not include alcoholic associated with granuloma formation and development of individuals and those with positive serology for HIV, HTLV- periportal fibrosis due to schistosomiasis, both in experi- 1, or hepatitis virus types B and C, all of which are conditions mental models and in vitro models of granuloma or tissue that could interfere with the immunological response. biopsies [9–11]. Recently described, the cytokine interleukin-17 (IL-17) 2.2. Ultrasound Examination. Abdominal ultrasound (USG) is considered as an inflammatory mediator and may be were performed using the Quantum 2000 Siemens and associated to the pathology of several chronic illnesses [12]. Elegra Siemens ultrasound with a convex transductor of 3.5– IL-17 stimulates the production of IL-6, nitric oxide, and 5.0 Mhz. Liver span was measured in the midclavicular line prostaglandin E2 (PGE2) and acts in synergy with other and midline. The liver was also examined for smoothness of α γ cytokines, such as IL-1, TNF- ,andIFN-. Furthermore, surface, echogenicity and posterior attenuation of the sound this cytokine promotes the proliferation and recruitment of bean, and portal vein diameter outside the liver midway monocytes and neutrophils to inflammatory sites. Th17 cells between its entrance into the portal hepatic and its first may have been involved in the pathogenesis of schistosomia- bifurcation in the liver. Periportal fibrosis was observed sis in experimental models [4, 13–15]. as multiple diffuse echogenic areas. Grading of periportal There are few studies evaluating the serum levels of fibrosis was determined by the mean total thickness of four cytokines and chemokines in schistosomiasis patients with portal tracts after the first division from the right and left ff di erent degrees of periportal fibrosis and the participation branches of portal vein (PT1) as follow: degree 0, mean of IL-17 in the formation of granuloma and progression thickness <3 mm; degree I, mean thickness 3 to 5 mm; degree to fibrosis in human schistosomiasis remains unclear. Thus, II, mean thickness >5 to 7 mm; and degree III mean thickness in this study apart from assessment of Th1, Th2, and T >7mm[20–22]. Of the 220 individuals evaluated 62 (28.2%) regulatory immune response we also evaluate the levels of IL- had some degree of periportal fibrosis as shown in Table 1. 17 in human schistosomiasis. The scores of periportal fibrosis were grouped according to the severity, being degree 0 without periportal fibrosis. Incipient periportal fibrosis was considered to individuals 2. Materials and Methods with degree I and moderate to severe periportal fibrosis to those with degrees II and III [23]. 2.1. Study Design and the Endemic Area. This study was To perform the immune response we included 30 indi- carried out in an endemic area from schistosomiasis named viduals with degree 0 and 25 individuals with degree I, while ´ ´ Agua Preta, in the state of Bahia, Brazil. Agua Preta is located all individuals with severe forms of the disease characterized 280 km south of Salvador, the capital of the State Bahia. It by the grade II (n = 13) or III (n = 04) were included. is composed of a residential area in the center of the village and some surrounding farms. A total of approximately 800 people live in the community. They live in poor sanitary 2.3. Schistosoma mansoni Antigens. The S. mansoni antigens conditions and agriculture is the predominant occupation. used in this study were the soluble extract of the adult worm There is one river in this region that is used for bathing, of S. mansoni (SWAP) and the soluble egg antigen (SEA), washing clothes and utensils, and leisure, exposing the prepared as previously described [24]. residents to high risk of Schistosoma infection. A cross-sectional parasitological surveys using Kato-Katz 2.4. Cell Culture and Cytokine Measurements. Peripheral [16] and sedimentation techniques were conducted in three blood mononuclear cells (PBMCs) were isolated using Ficoll- different stool samples collected on different days. The cur- Hypaque gradient sedimentation and adjusted to a concen- rent degree of exposure to S. mansoni infection was assessed tration of 3 × 106/mL in RPMI 1640 medium containing by a previously developed questionnaire, which provides 10% normal human serum (AB positive and heat inacti- four categories of reported level of exposure to infested vated), 100 U/mL of penicillin, 100 mg/mL of streptomycin, water: no exposure, low exposure (<1 h/week), medium 2 mmol/L of L-glutamine, and 30 mmol/L of HEPES (all exposure (1–3 h/week), or high exposure (1–3 h/day) [17– from Life Technologies GIBCO, BRL, Gaithersburg, MS). 19]. Cells were cultured without any stimulation or stimulated Journal of Parasitology Research 3

Table 1: Demographic characteristics of the individuals enrolled in the study (n = 220).

Characteristic Without fibrosis (n = 158) Incipient fibrosis (n = 45) Moderate to severe fibrosis (n = 17) P Age (median/range)∗ 19 (5–60) 30 (9–58) 40 (21–59) <0.001a,b Gender n (%)∗∗ Male 83 (52.5) 23 (51.1) 8 (47.1) ns Female 75 (47.5) 22 (48.9) 9 (52.9) Burden parasite for Schistosoma mansoni (EPG/range)∗ 72 (24–4752) 42 (24–1380) 60 (24–200) 0.0451a Coinfection (Schistosoma mansoni and other helminth infections) (%)∗∗ Yes 74.7 68.3 66.7 ns No 25.3 31.7 33.3 ∗Kruskal-Wallis; ∗∗Chi-squared Test; awithout fibrosis versus incipient fibrosis; bwithout fibrosis versus moderate to severe fibrosis. EPG: eggs per gram of feces; ns: not significant (P>0.05). with 10 mg/mL of SWAP, SEA, or Phytohemagglutinin with incipient and moderate to severe periportal fibrosis was (PHA). Plates were incubated at 37◦Cinanatmosphere higher than in individuals without fibrosis (P<0.05). containing 5% CO2. Supernatants were collected after 72 There was no significant difference in gender distribution hours of incubation and maintained at −20◦Cformeasure- among groups. There was also no significant difference ment of cytokines by Enzyme-linked immunosorbent assay in the levels of exposure to the contaminated water, with (ELISA). Levels of IL-5, IL-13, IL-17, IL-10, IFN-γ,andTNF- medium to high contact to the water being found in α were determined (R&D Systems, Inc, Minneapolis, MN), 60.5%, 55.3%, and 80.0% of individuals without periportal and results were expressed as pg/mL on the basis of standard fibrosis, incipient fibrosis, and moderate to severe fibrosis, curves. respectively (P>0.05). The S. mansoni parasite burden of individuals without periportal fibrosis was higher than in patients with incipient 2.5. Serum Cytokine and Chemokine Measurement. Levels of fibrosis (P<0.05). There was no statistically significant the cytokines IL-5, IL-13, IFN-γ,TNF-α, IL-17, IL-10, TGF-β difference of being coinfected with other helminths in all (R&D systems Inc., Minneapolis) and the chemokines MIP- groups of patients (Table 1). 1α/CCL3 and RANTES/CCL5 were measured in serum using sandwich ELISA (eBioscience). The results are expressed as pg/mL, based in standard curves. 3.2. Cytokine Responses Induced by S. mansoni Antigens. Levels of IL-5, IL-13, IL-17, IFN-γ,TNF-α, and IL-10 were measured in supernatant of PBMCs cultures unstimulated 2.6. Statistical Analysis. Statistical analyses were performed and stimulated with SWAP and SEA as shown in Table 2.A using the Statistical Package for the Social Sciences software significantly higher levels of IL-5 in cultures stimulated with (version 9.0 for Windows, SPSS). Statistical differences SEA was observed in the group of patients with moderate between groups were assessed using the Kruskal-Wallis to severe fibrosis (median levels = 310.9 pg/mL) compared analysis of variance test. Fisher’s exact test was used to to those without fibrosis (median levels = 36.8 pg/mL; P = compare proportions. To assess the association between the 0.0418). serum levels of cytokines and chemokines and the parasite Levels of TNF-α were significantly higher in supernatants burden of S. mansoni the Linear Regression tests using from SWAP-stimulated PBMC of subjects with incipient Graphpad PRISM 3.03 software (La Jolla, CA, USA) were periportal fibrosis (median levels = 1446 pg/mL) than in used. All statistical tests were two-tailed and the statistical individuals without fibroses (median levels = 756.1 pg/mL; significance was established at the 95 percent confidence P = 0.0319). Moreover, the levels of TNF-α in nonstimulated interval and significance was defined to P<0.05. cultures were higher in individuals with moderate to severe The Ethical Committee of Climerio´ de Oliveira Mater- fibrosis (median levels = 488.7 pg/mL) compared to subjects nity, Federal University of Bahia, approved the present study. without periportal fibrosis (median levels = 61.9 pg/mL; P = Informed consent was obtained from all study participants 0.0182). or their legal guardians (License number 240/2008). The levels of IL-13, IL-17, IFN-γ, and IL-10 did not differ significantly between individuals of the different groups 3. Results (Table 2). 3.1. Features of the Studied Subject. The demographic 3.3. Serum Levels of Cytokines. The levels of serum cytokines characteristics, parasite burden, and the ultrasonography in patients with different degrees of periportal fibrosis are evaluation for periportal fibrosis measurement are shown in shown in Figure 1. With the exception of IFN-γ and IL-17, Table 1. It was observed that the mean age of individuals whose levels were below the detection limit of 15.6 pg/mL 4 Journal of Parasitology Research

Table 2: Levels of cytokines produced by peripheral blood mononuclear cells (PBMC) of studied individuals.

Cytocines (pg/mL) Without fibrosis Incipient fibrosis Moderate to severe P median (min–max) (n = 30) (n = 25) Fibrosis (n = 17) IL-5 Without antigen 31.2 (31.2–112.2) 31.2 (31.2–75.5) 31.2 (31.2–127.8) ns SWAP 374.4 (31.2–5153.0) 524 (31.2–5905.0) 1988.0 (31.2–5135.0) ns SEA 36.8 (31.2–3820.0) 188.4 (31.2–4232.0) 310.9 (31.2–4256.0) 0.0418a IL-13 Without antigen 112.4 (93.8–186.8) 93.8 (93.8–166.0) 93.8 (93.8–93.8) ns SWAP 246.6 (93.8–1518.0) 190.3 (93.8 –1290.0) 213.7 (93.8–2091.0) ns SEA 171.2 (93.8–1497.0) 93.8 (93.8–1442.0) 93.8 (93.8–1857.0) ns IL-17 Without antigen 15.6 (15.6–132.9) 15.6 (15.6–84.1) 15.6 (15.6–90.7) ns SWAP 21.4 (15.6–790.5) 57.1 (15.6–974.8) 21.4 (15.6–345.2) ns SEA 18.9 (15.6–291.1) 15.6 (15.6–615.3) 27.2 (15.6–557.5) ns IFN-γ Without antigen 31.2 (31.2–276.2) 31.9 (31.2–1788.0) 31.2 (31.2–777.7) ns SWAP 73.1 (31.2–3038.0) 1205.0 (31.2–5507.0) 215.8 (31.2–5249.0) ns SEA 83.1 (31.2–288.0) 203.8 (31.2–2905.0) 130.1 (31.2–2714.0) ns TNF-α Without antigen 61.9 (31.2–633.3) 346.6 (31.2–2421.0) 488.7 (31.2–3229.0) 0.0182a SWAP 756.1 (80.6–3366.0) 1446.0 (236.4–3057.0) 1002.0 (35.7–2351.0) 0.0319b SEA 389.8 (102.7–2521.0) 827.3 (31.2–3551.0) 927.3 (89.1–2517.0) ns IL-10 Without antigen 15.6 (15.6–705.5) 48.6 (15.6–1486.0) 17.5 (15.6–1262.0) ns SWAP 393.8 (22.1–1976.0) 399.6 (15.6–1779.0) 318.1 (15.6–1213.0) ns SEA 585.7 (175.7–2075.0) 1342.0 (15.6–2472.0) 745.8 (35.5–1543.0) ns ∗Kruskal-Wallis; awithout fibrosis versus moderate to severe fibrosis; bwithout fibrosis versus incipient fibrosis; ns: not significant (P>0.05). SWAP: soluble worm antigen preparation of adult Schistosoma mansoni;SEA:Schistosoma mansoni egg antigen. and 31.2 pg/mL, respectively, in the three groups of patients levels of IL-13, TNF-α,MIP-1α, and RANTES and parasite (data not shown), high levels of serum cytokines were burden was observed (Figure 3). There were no significant observed in the different groups of individuals (Figure 1). associations between serum levels of IL-5 (R2 = 0.003413; While the median levels of IL-13 were higher in indi- P>0.05), IFN-γ (R2 = 0.005831; P>0.05), IL-10 (R2 = viduals without periportal fibrosis compared to those with 0.004651; P>0.05), and TGF-β (R2 = 0.003391; P>0.05) moderate to severe fibrosis (113.3 pg/mL and 97.7 pg/mL, and the parasite burden of S. mansoni. respectively; P = 0.0006), there was no significant difference in the median levels of IL-5, IL-10, TNF-α and TGF-β between groups (P>0.05; Figure 1). 4. Discussion The development of periportal fibrosis can occur as a result 3.4. Serum Level of the Chemokines . The median levels of the of chronic schistosomiasis infection and accounts for the chemokines MIP-1α and RANTES in serum of individuals severe forms of the disease [6]. This fact characterizes with different degrees of periportal fibrosis are shown in schistosomiasis as a serious public health problem. Figure 2. It was observed that the median level of MIP- The present study aimed to evaluate cytokine and 1α was higher in individuals without periportal fibrosis chemokine profile in individuals with different degrees of (70.9 pg/mL) compared to those with moderate to severe periportal fibrosis living in the schistosomiasis endemic fibrosis (7.8 pg/mL; P = 0.0232). There was no significant area of Agua´ Preta, Brazil. Additionally, possible personal difference in serum levels of RANTES in individuals with and environmental features which could interfere with the different degrees of periportal fibrosis. development of periportal fibrosis were also evaluated. We showed that despite the high prevalence of S. mansoni 3.5. Association between Serum Levels of Cytokines and infection in Agua´ Preta, Bahia, the frequency of severe Chemokines and S. mansoni Parasite Burden. We tested forms of the disease determined by ultrasonography was the possible association between serum levels of cytokines low. This could be due to intrinsic characteristics of this and chemokines and S. mansoni parasite burden in the population, since there is no report of previous treatment studied population. A positive association between serum for schistosomiasis in this region. Other possible explanation Journal of Parasitology Research 5

IL-5 IL-13 800 400 ∗

200 200 200 200

150 150 IL-5 (pg/mL) IL-5 IL-13 (pg/mL) IL-13 100 100

50 50

0 0 Without ﬁbrosis Incipient Moderate to severe Without ﬁbrosis Incipient Moderate to severe (a) (b) 800 8000 TGF-β TNF-α

6000

200

200 (pg/mL) (pg/mL) β 4000 α 150 TGF- TNF- 100 2000 50

0 0 Without ﬁbrosis Incipient Moderate to severe Without ﬁbrosis Incipient Moderate to severe (c) (d)

800 IL-10

300 300

IL-10 (pg/mL) IL-10 200

100

0 Without ﬁbrosis Incipient Moderate to severe (e)

Figure 1: Serum cytokine levels in schistosomiasis individuals with different degrees of periportal fibrosis: (first box) individuals without fibrosis (n = 30), (second box) incipient fibrosis (n = 25), and (third box) moderate to severe periportal fibrosis (n = 17). Levels of IL-5, IL- 13, TNF-α,TGF-β, and IL-10 were determined by sandwich ELISA technique. Horizontal lines represent the median values, boxes represent the 25th to the 75th percentiles, and vertical lines represent the 10th to 90th percentiles. Asterisks indicate statistically significant differences (P<0.05; ANOVA).

would be the Cairo’s methodology to classify the periportal The majority of individuals who had the most severe fibrosis which considers only degrees II and III as severe. degree of periportal fibrosis were over 40 years of age, and We opted to use the Cairo’s classification because we have there was no influence of gender on periportal fibrosis devel- performed previous studies using these parameters and opment. This is in agreement with other authors who have because the physicians who have performed the USG in our demonstrated that most individuals who develop periportal studies are very well familiar with this classification. fibrosisareover50yearsofage[22]. This could be explained 6 Journal of Parasitology Research

MIP-1α ∗ 400 1100 RANTES

1050 300

1000 /CCL3 α 200 950 MIP-1

100 RANTES/CCL5 900

0 850 Without ﬁbrosis Incipient Moderate to severe Without ﬁbrosis Incipient Moderate to severe (a) (b)

Figure 2: Serum chemokine levels in schistosomiasis individuals with different degrees of periportal fibrosis: (first box) individuals without fibrosis (n = 30), (second box) incipient fibrosis (n = 25), and (third box) moderate to severe periportal fibrosis (n = 17). Levels of MIP-1α and RANTES were determined by sandwich ELISA technique. Horizontal lines represent the median values, boxes represent the 25th to the 75th percentiles, and vertical lines represent the 10th to 90th percentiles. Asterisks indicate statistically significant differences (P<0.05; ANOVA).

2000 500 α IL-13 TNF- 400 ∗R2 = 0.06642 ∗R2 = 0.1624 P<0.05 P<0.05 500 300

500 (pg/mL) α 200 IL-13 (pg/mL) IL-13 TNF- 250 100

0 0 0 250 500 750 1000 1250 0 250 500 750 1000 1250 EPG of feces EPG of feces (a) (b) 1200 1000 RANTES ∗R2 = 0.0713 MIP-1α ∗ P<0.05 750 R2 = 0.3183 1100 P<0.05 (pg/mL) 500 1000 α RANTES (pg/mL)

MIP-1 250 900 900 0 0 0 250 500 750 1000 1250 0 250 500 750 1000 1250 EPG of feces EPG of feces (c) (d)

Figure 3: Correlation between serum levels of IL-13, TNF-α,MIP-1α, and RANTES (n = 65) and S. mansoni parasite burden. P<0.05 indicates statistically significant differences (Linear Regression). Journal of Parasitology Research 7 by the immune response induced by constant reexposure to found, however, higher levels of serum IL-13 in the group of the parasite over lifetime, or by the slow process of fibrosis individuals without periportal fibrosis compared to patients formation. Therefore, younger individuals probably have with severe fibrosis. It has been suggested that levels of IL- not been exposed long enough to the cumulative effects of 13 tend to be higher during the prefibrotic phase, acting as collagen deposition in the periportal tract [25]. an inducer of the lesion [35, 39, 40]. The parasite burden is Many variables may influence the magnitude of the one of many other variables that can influence the magnitude immune response in human schistosomiasis which includes of the immune response in human schistosomiasis [32, 41] gender and age [26], intensity of infection [27–30], and and a high parasite burden has been associated with the genetic characteristics of the population [31]. However, the development of fibrosis [5, 6]. Corroborating with these reason why severe fibrosis develops only in a fraction of observation, we also found a positive association between the population, which is under the same environmental parasite burden and serum levels of IL-13. conditions as others who do not develop fibrosis, remains There are no published data about the role of IL-17 in not well understood. In this study, we found no association development of hepatic fibrosis due to schistosomiasis in between level of exposure to contaminated water and degree humans. As reviewed byTallima et al. (2009), this cytokine of periportal fibrosis. Moreover, individuals without peripor- is involved in the development of severe disease by recruit- tal fibrosis had a higher parasite burden than individuals with ment of neutrophils and macrophages in inflammatory incipient periportal fibrosis. A possible explanation for this sites, demonstrated only in experimental models [42]. We observation is that chronic infection can lead to intestinal observed production of IL-17 in cultures stimulated by all fibrosis that impairs the migration of eggs to the intestinal the antigens tested without, however, differences among lumen and thereby decreases eggs count in parasitological the groups. It is suggested that induction of severe liver exams [32]. disease associated with expression of IL-17 in experimental It has been described that the specific type 2 cytokine models, with polarized immune response to a Th1 profile, is pattern is a marker of S. mansoni chronic infection in dependent on the presence of IL-23, another Th17 cytokine mice and humans, and that this response is involved in the [12, 13]. In this study, we evaluated the IL-17 production development of periportal fibrosis due to schistosomiasis in individuals chronically infected by S. mansoni,who [21, 33–35]. In this study we evaluated cytokine production have a predominantly Th2 immune response, which may by PBMC stimulated with the S. mansoni antigens SWAP and explain the low and not detectable levels of IL-17. When SEA as well as its serum levels and correlation with parasite evaluating serum levels of this cytokine we also found levels load. We observed a greater variability in cytokines levels, of IL-17 below the detection limit in most individuals, which shows heterogeneity of response to the S. mansoni which corroborates the results observed when measuring the antigens. supernatants of PBMCs cultures. Moreover, there was no Levels of IL-5 in supernatants of SEA-stimulated PBMCs correlation between serum levels of IL-17 and S. mansoni were higher in cultures of individuals with moderate to parasite load. severe periportal fibrosis, when compared to the group Some authors have suggested that IFN-γ has certain without fibrosis. These data are similar to what was observed antifibrotic activities, since this cytokine acts by inhibiting in a study conducted in schistosomiasis patient’s residents in production of extracellular matrix proteins, enhances the other endemic areas of Bahia [36, 37]. These data suggest activity of collagenase in liver tissue, and downmodulates that the Th2 immune response is developedand directed the Th2 response [10, 43]. In this study, we found no to antigens from the eggs. IL-5 has been associated with significant differences in levels of IFN-γ between groups, granuloma formation around S. mansoni eggs in the liver, and no significant association between parasite burden and since this cytokine participates in eosinophil growth and serum levels of this cytokine in individuals with different activation contributing to a significant source of profibrotic levels of periportal fibrosis. mediators such as IL-13 [38]. Likewise, IL-5 may participate Although controversy, TNF-α is another cytokine that directly in tissue remodeling and fibrosis in schistosomiasis may participate in the granuloma formation and evolution of [38]. In contrast to our data that showed no significant fibrotic tissue process.Hoffmann and colleagues (1998) have difference or correlation between serum levels of IL-5 and S. demonstrated in experimental models that TNF-α exerts mansoni parasite burden in individuals with different degrees aprotectiveeffect, whereas other authors attribute to the of periportal fibrosis, it has been described that serum levels TNF-α proinflammatory and profibrogenic effects [7, 44]. of IL-5 is higher in patients with severe degrees of periportal In the present study it was observed that cells of individuals fibrosis than in those without fibrosis [39]. with incipient or moderate to severe fibrosis, even without Many studies in experimental models have shown that antigenic stimuli, produced higher levels of TNF-α when IL-13 plays an important role in the development of liver compared to those without fibrosis. Additionally, there fibrosis. The blockage of IL-13 and IL-4 receptors prevented was a positive association between serum levels of TNF-α granuloma development and liver fibrosis in mice [33]. and S. mansoni parasite burden, which suggests that this Human studies suggest a correlation between production cytokine may contribute to the liver pathology observed in of high levels of IL-13 and development of more advanced schistosomiasis [45]. Supporting the role of TNF-α in the degree of liver fibrosis [36, 37]. However, in this study we development of liver pathology due to schistosomiasis, a did not observe differences in IL-13 levels in supernatants of study conducted in a schistosomiasis endemic area in Brazil SWAP or SEA stimulated PBMCs in all groups evaluated. We has demonstrated that individuals with moderate to severe 8 Journal of Parasitology Research periportal fibrosis have higher serum levels of TNF-α than RANTES, whereas Th2 cytokines, such as IL-4 and IL-13, are those without fibrosis [39]. associated with decreased expression [54–56]. In agreement Some studies have pointed out TGF-β as an important with these studies, it has shown that RANTES deficient mice factor in periportal fibrosis development during chronic showed a significant increase granulomatous response when schistosomiasis. This cytokine is considered a multifunc- compared to the control group [57]. These studies reinforce tional cytokine that regulates biological processes such as the idea that RANTES regulates negatively the development inflammation, development, and differentiation of many cell of granuloma and fibrosis. On the other hand, there are no types, tissue repair, and tumor genesis. It is also associated data in the literature on the possible role of RANTES in the with pro-inflammatory responses and immunosuppressive development of periportal fibrosis in human schistosomiasis. activities [46, 47] and participates in the process of Th17 cells The present study showed high levels of IL-5 and TNF-α differentiation [42]. In this study, we found no significant in individuals with periportal fibrosis. Higher levels of IL-13 differences in serum levels of TGF-β between groups with and MIP-1α in individuals without periportal fibrosis were different degrees of periportal fibrosis, nor did we find an also documented. Considering that these former molecules association between parasite burden and levels of TGF-β. were positively associated with S. mansoni parasite burden, as Recent studies in experimental models have shown a negative were TNF-α and RANTES, they may represent biomarker for association between serum levels of TGF-β and S. mansoni the progression of liver pathology in schistosomiasis. Larger parasite burden in chronically infected animals, suggesting casuistic further studies are needed however to confirm the the involvement of this cytokine in controlling the parasite role of these cytokines and chemokines in the development load [48]. of periportal fibrosis in human schistosomiasis. Other molecule assessed in this study was the regulatory cytokine IL-10, which is associated with the switch from the Th1 immune response observed during the acute phase to Acknowledgments Th2 responses and consequently preventing the development The authors would like to thanks to Dr. Marta Leite and Dr. of severe disease. In this study we found no significant Antonio Carlos M. Lemos for their support in the selection ff di erence in the serum levels of this cytokine,nor in the of patients, and Dr. Irisma´ Souza and Dr. Delfin Gonzalez for levels measured in supernatant of stimulated cultures among performing the ultrasound assessment. they are very grateful ff individuals with di erent degrees of periportal fibrosis. to all volunteers from the community of Agua´ Preta, Gandu, This is in agreement with Silva-Teixeira and coworkers who agreed to partipate in this study, to the local health agent (2004) who demonstrated no association between levels Irene Jesus for her support, and to Michael Andrew Sundberg of IL-10 and parasite burden, suggesting that IL-10 may for the corrections and suggestions made in the text. They not be involved in periportal fibrosis development during also thank the Fundaçao˜ de Amparo a` Pesquisa do Estado schistosomiasis [39]. da (Bahia FAPESB) for the financial support. E. Carvalho Levels of cytokines in patients with schistosomiasis have andM.I.AraujoareinvestigatorssupportedbyTheCon- been evaluated in serum or supernatants of cell cultures. In selho Nacional de Desenvolvimento Cient´ıfico e Tecnologico´ this study we decided to evaluate cytokine in both, serum (CNPq). This work was supported by the Brazilian National and supernatants. We believe that serum cytokine levels Research Council (CNPq) Universal (482113/2010-3). represent the in vivo profile status. However, as paracrine cytokines, such as IL-5, IL-13, IFN-γ, and IL-10, are not well detected in serum using convenient technique such as ELISA, References we decided to evaluate these molecules in supernatants of PBMC restimulated in vitro with parasite antigens. We [1] L. S. Iarotski and A. Davis, “The schistosomiasis problem in the world: results of a WHO questionnaire survey,” Bulletin believe that these two measurements are complementary and of the World Health Organization, vol. 59, no. 1, pp. 115–127, lead to a better knowledge of the cytokine profile in human 1981. schistosomiasis. [2] Ministerio´ da Saude,´ Guia de Vigilanciaˆ Epidemiologica´ , Min- The role of chemokines in periportal fibrosis due to isterio´ da Saude,´ Bras´ılia, Brasil, 2005. schistosomiasis is also not completely understood [49]. In [3] M. S. Wilson, M. M. Mentink-Kane, J. T. Pesce, T. R. Rama- this study, we found higher levels of serum MIP-1α in lingam, R. Thompson, and T. A. Wynn, “Immunopathology individuals without periportal fibrosis than in patients with of schistosomiasis,” Immunology and Cell Biology, vol. 85, no. incipient or moderate to severe fibrosis, which is in contrast 2, pp. 148–154, 2007. to what has been found by Souza and colleagues [50, 51]. We [4] Z. A. Andrade, “Schistosomiasis and liver fibrosis: review showed, however, a positive association between serum levels Article,” Parasite Immunology, vol. 31, no. 11, pp. 656–663, of MIP-1α and S. mansoni parasite burden. This finding is 2009. similar to what was obtained for IL-13 and reinforces the [5]J.C.Bina,“Estudodevariaveis´ que podem influenciar na evoluçao˜ da esquistossomose mansonica:ˆ efeito da terapeuticaˆ thought that individuals without fibrosis enrolled in this espec´ıfica e da interrupçao˜ da transmissao,”` Revista de Patolo- study may have been in a prefibrotic stage. Interestingly, it α gia Tropical, vol. 26, pp. 69–128, 1997. has been also demonstrated that MIP-1 induces production [6] J. C. Bina and A. Prata, “Schistosomiasis in hyperendemic area of Th2-pattern cytokines, including IL-13 [52, 53]. of Taquarendi. I- Schistosoma mansoni infection and severe Many authors have suggested that high levels of IFN- clinikal forms,” Revista da Sociedade Brasileira de Medicina γ and TNF-α are associated with increased expression of Tropical, vol. 36, no. 2, pp. 211–216, 2003. Journal of Parasitology Research 9

[7] S. Henri, C. Chevillard, A. Mergani et al., “Cytokine regulation [22] L. F. A. Oliveira, E. C. Moreno, G. Gazzinelli et al., “Cytokine of periportal fibrosis in humans infected with Schistosoma production associated with periportal fibrosis during chronic mansoni:IFN-γ is associated with protection against fibrosis schistosomiasis mansoni in humans,” Infection and Immunity, and TNF-α with aggravation of disease,” Journal of Immunol- vol. 74, no. 2, pp. 1215–1221, 2006. ogy, vol. 169, no. 2, pp. 929–936, 2002. [23] E. J. Pearce, S. L. James, and J. Dalton, “Immunochemical [8]P.L.Falcao,˜ L. C. C. Malaquias, O. A. Martins-Filho et al., characterization and purification of Sm-97, a Schistosoma “Human Schistosomiasis mansoni: IL-10 modulates the in mansoni antigen monospecifically recognized by antibodies vitro granuloma formation,” Parasite Immunology, vol. 20, no. from mice protectively immunized with a nonliving vaccine,” 10, pp. 447–454, 1998. Journal of Immunology, vol. 137, no. 11, pp. 3593–3600, 1986. [9] S. Gustavson, C. S. Zouain, J. B. Alves, M. F. Leite, and [24] C. Hirsch and A. M. Goes, “Characterization of fractionated A. M. Goes, “Modulation of granulomatous hypersensitivity Schistosoma mansoni soluble adult worm antigens that elicit against Schistosoma mansoni eggs in mice vaccinated with human cell proliferation and granuloma formation in vitro,” culture-derived macrophages loaded with PIII,” Parasitology Parasitology, vol. 112, no. 6, pp. 529–535, 1996. International, vol. 51, no. 3, pp. 259–269, 2002. [25] M. Booth, J. K. Mwatha, S. Joseph et al., “Periportal Fibrosis in [10] T. A. Wynn, “Cytokine regulation of granuloma formation in Human Schistosoma mansoni Infection Is Associated with Low schistosomiasis,” Current Opinion in Immunology, vol. 7, no. IL-10, Low IFN-γ, High TNF-α, or Low RANTES, Depending 4, pp. 505–511, 1995. on Age and Gender,” Journal of Immunology, vol. 172, no. 2, [11] L. Steinman, “A brief history of TH17, the first major pp. 1295–1303, 2004. revision in the T H1/TH2 hypothesis of T cell-mediated tissue [26] Q. Mohamed-Ali, N. E. M. A. Elwali, A. A. Abdelhameed et al., damage,” Nature Medicine, vol. 13, no. 2, pp. 139–145, 2007. “Susceptibility to periportal (Symmers) fibrosis in human [12] L. I. Rutitzky, L. Bazzone, M. G. Shainheit, B. Joyce-Shaikh, Schistosoma mansoni infections: evidence that intensity and D. J. Cua, and M. J. Stadecker, “IL-23 is required for the duration of infection, gender, and inherited factors are critical development of severe egg-induced immunopathology in in disease progression,” Journal of Infectious Diseases, vol. 180, schistosomiasis and for lesional expression of IL-17,” Journal no. 4, pp. 1298–1306, 1999. of Immunology, vol. 180, no. 4, pp. 2486–2495, 2008. [27] M. F. Abdel-Wahab, G. Esmat, S. I. Narooz, A. Yosery, J. [13]L.I.Rutitzky,J.R.L.DaRosa,andM.J.Stadecker, P. Struewing, and G. T. Strickland, “Sonographic studies of “Severe CD4 T cell-mediated immunopathology in murine schoolchildren in a village endemic for Schistosoma mansoni,” schistosomiasis is dependent on IL-12p40 and correlates with Transactions of the Royal Society of Tropical Medicine and high levels of IL-17,” Journal of Immunology, vol. 175, no. 6, Hygiene, vol. 84, no. 1, pp. 69–73, 1990. pp. 3920–3926, 2005. [28] E. Doehring-Schwerdtfeger, I. M. Abdel-Rahim, Q. [14] L. I. Rutitzky and M. J. Stadecker, “CD4 T cells producing Mohamed-Ali et al., “Ultrasonographical investigation pro-inflammatory interleukin-17 mediate high pathology in of peripheral fibrosis in children with Schistosoma mansoni schistosomiasis,” Memorias do Instituto Oswaldo Cruz, vol. infection: evaluation of morbidity,” American Journal of 101, no. 1, pp. 327–330, 2006. Tropical Medicine and Hygiene, vol. 42, no. 6, pp. 581–586, [15]N.Katz,P.M.Coelho,andJ.Pellegrino,“EvaluationofKato’s 1990. quantitative method through the recovery of Schistosoma mansoni eggsaddedtohumanfeces,”Journal of Parasitology, [29] A. L. C. Domingues, A. R. F. Lima, H. S. Dias, G. C. Leao, vol. 56, no. 5, pp. 1032–1033, 1970. and A. Coutinho, “An ultrasonographic study of liver fibrosis [16] M. I. A. S. Araujo, B. Hoppe, M. Medeiros et al., “Impaired in patients infected with Schistosoma mansoni in north-east T helper 2 response to aeroallergen in helminth-infected Brazil,” Transactions of the Royal Society of Tropical Medicine patiente with asthma,” Journal of Infectious Diseases, vol. 190, and Hygiene, vol. 87, no. 5, pp. 555–558, 1993. no. 10, pp. 1797–1803, 2004. [30]M.Homeida,A.F.Abdel-Gadir,A.W.Cheeveretal., [17] A. J. Dessein, M. Begley, C. Demeure et al., “Human resistance “Diagnosis of pathologically confirmed Symmers’ periportal to Schistosoma mansoni is associated with IgG reactivity to a fibrosis by ultrasonography: a prospective blinded study,” 37-kDa larval surface antigen,” Journal of Immunology, vol. American Journal of Tropical Medicine and Hygiene, vol. 38, no. 140, no. 8, pp. 2727–2736, 1988. 1, pp. 86–91, 1988. [18] A. V. Grant, M. I. Araujo, E. V. Ponte et al., “High heri- [31]A.J.Dessein,D.Hillaire,N.E.M.A.Elwalietal.,“Severe tability but uncertain mode of inheritance for total serum hepatic fibrosis in Schistosoma mansoni infection is controlled γ IgE level and Schistosoma mansoni infection intensity in by a major locus that is closely linked to the interferon- a schistosomiasis-endemic Brazilian population,” Journal of receptor gene,” American Journal of Human Genetics, vol. 65, Infectious Diseases, vol. 198, no. 8, pp. 1227–1236, 2008. no. 3, pp. 709–721, 1999. [19]M.F.Abdel-Wahab,G.Esmat,A.Farrag,Y.A.El-Boraey,and [32]O.S.Carvalho,P.M.Z.Coelho,andH.L.Lenzi,Schistosoma G. T. Strickland, “Grading of hepatic schistosomiasis by the mansoni e Esquistossomose: Uma Visao˜ Multidisciplinar, Edi- use of ultrasonography,” American Journal of Tropical Medicine tora Fiocruz, Rio de Janeiro, Brazil, 1st edition, 2008. and Hygiene, vol. 46, no. 4, pp. 403–408, 1992. [33] M. G. Chiaramonte, A. W. Cheever, J. D. Malley, D. D. Don- [20] A. R. De Jesus, A. Silva, L. B. Santana et al., “Clinical and aldson, and T. A. Wynn, “Studies of murine schistosomiasis immunologic evaluation of 31 patients with acute schistoso- reveal interleukin-13 blockade as a treatment for established miasis mansoni,” Journal of Infectious Diseases, vol. 185, no. 1, and progressive liver fibrosis,” Hepatology, vol. 34, no. 2, pp. pp. 98–105, 2002. 273–282, 2001. [21] A. R. De Jesus, D. G. Miranda, R. G. Miranda et al., “Morbidity [34] M. G. Chiaramonte, D. D. Donaldson, A. W. Cheever, and T. A. associated with Schistosoma mansoni infection determined by Wynn, “An IL-13 inhibitor blocks the development of hepatic ultrasound in an endemic area of Brazil, Caatinga do Moura,” fibrosis during a T-helper type 2-dominated inflammatory American Journal of Tropical Medicine and Hygiene, vol. 63, no. response,” Journal of Clinical Investigation, vol. 104, no. 6, pp. 1-2, pp. 1–4, 2000. 777–785, 1999. 10 Journal of Parasitology Research

[35] A. R. De Jesus, I. Araujo,´ O. Bacellar et al., “Human immune [50] A. L. S. Souza, E. Roffe,ˆ V. Pinho et al., “Potential role of the responses to Schistosoma mansoni vaccine candidate antigens,” chemokine macrophage inflammatory protein 1α in human Infection and Immunity, vol. 68, no. 5, pp. 2797–2803, 2000. and experimental schistosomiasis,” Infection and Immunity, [36] A. R. De Jesus, A. Magalhaes,˜ D. G. Miranda et al., “Association vol. 73, no. 4, pp. 2515–2523, 2005. of type 2 cytokines with hepatic fibrosis in human Schistosoma [51] P. R. S. Souza, A. L. S. Souza, D. Negrao-Correa,˜ A. L. mansoni infection,” Infection and Immunity,vol.72,no.6,pp. Teixeira, and M. M. Teixeira, “The role of chemokines 3391–3397, 2004. in controlling granulomatous inflammation in Schistosoma [37] A. Magalhaes,˜ D. G. Miranda, R. G. Miranda et al., “Cytokine mansoni infection,” Acta Tropica, vol. 108, no. 2-3, pp. 135– profile associated with human chronic schistosomiasis man- 138, 2008. soni,” Memorias do Instituto Oswaldo Cruz, vol. 99, no. 5, [52] P. L. Falcao,˜ R. Correa-Oliveira, L. A. O. Fraga et al., “Plasma supplement 1, pp. 21–26, 2004. concentrations and role of macrophage inflammatory protein- [38] R. M. Reiman, R. W. Thompson, C. G. Feng et al., 1α during chronic Schistosoma mansoni infection in humans,” “Interleukin-5 (IL-5) augments the progression of liver fibro- Journal of Infectious Diseases, vol. 186, no. 11, pp. 1696–1700, sis by regulating IL-13 activity,” Infection and Immunity, vol. 2002. 74, no. 3, pp. 1471–1479, 2006. [53] D. M. Oliveira, D. N. Silva-Teixeira, S. Gustavson, S. M. P. [39] D. N. Silva-Teixeira, C. Contigli, J. R. Lambertucci, J. C. Serufo, Oliveira, and A. M. Goes, “Nitric oxide interaction with IL- and V. Rodrigues, “Gender-related cytokine patterns in sera of 10, MIP-1α, MCP-1 and RANTES over the in vitro granuloma schistosomiasis patients with symmers’ fibrosis,” Clinical and formation against different Schistosoma mansoni antigenic Diagnostic Laboratory Immunology, vol. 11, no. 3, pp. 627–630, preparations on human schistosomiasis,” Parasitology, vol. 2004. 120, no. 4, pp. 391–398, 2000. [40]C.N.L.DeMorais,J.R.DeSouza,W.G.DeMeloetal., [54] N. Berkman, V. L. Krishnan, T. Gilbey et al., “Expression of “Studies on the production and regulation of interleukin, IL- RANTES mRNA and protein in airways of patients with mild 13, IL-4 and interferon-γ in human schistosomiasis mansoni,” asthma,” American Journal of Respiratory and Critical Care Memorias do Instituto Oswaldo Cruz, vol. 97, supplement 1, pp. Medicine, vol. 154, no. 6, pp. 1804–1811, 1996. 113–114, 2002. [55] M. John, S. J. Hirst, P. J. Jose et al., “Human Airway smooth [41] M. I. Araujo,´ A. R. De Jesus, O. Bacellar, E. Sabin, E. Pearce, muscle cells express and release RANTES in response to T and E. M. Carvalho, “Evidence of a T helper type 2 activation helper 1 Cytokines: regulation by T helper 2 cytokines and in human schistosomiasis,” European Journal of Immunology, corticosteroids,” Journal of Immunology, vol. 158, no. 4, pp. vol. 26, no. 6, pp. 1399–1403, 1996. 1841–1847, 1997. [42] H. Tallima, M. Salah, F. R. Guirguis, and R. El Ridi, “Trans- [56] A. Marfaing-Koka, O. Devergne, G. Gorgone et al., “Reg- forming growth factor-β and Th17 responses in resistance to ulation of the production of the RANTES chemokine by primary murine schistosomiasis mansoni,” Cytokine, vol. 48, endothelial cells: synergistic induction by IFN-γ plus TNF-α no. 3, pp. 239–245, 2009. and inhibition by IL-4 and IL-13,” Journal of Immunology, vol. [43] L. C. Borish and J. W. Steinke, “2. Cytokines and chemokines,” 154, no. 4, pp. 1870–1878, 1995. Journal of Allergy and Clinical Immunology, vol. 111, no. 2, [57] S. W. Chensue, K. S. Warmington, E. J. Allenspach et al., supplement, pp. S460–S475, 2003. “Differential expression and cross-regulatory function of [44] K. F. Hoffmann, P. Caspar, A. W. Cheever, and T. A. Wynn, RANTES during mycobacterial (type 1) and schistosomal “IFN-γ, IL-12, and TNF-α are required to maintain reduced (type 2) antigen-elicited granulomatous inflammation,” Jour- liver pathology in mice vaccinated with Schistosoma mansoni nal of Immunology, vol. 163, no. 1, pp. 165–173, 1999. eggs and IL-12,” Journal of Immunology, vol. 161, no. 8, pp. 4201–4210, 1998. [45]S.A.Shahat,M.A.El-Dhshan,S.S.Aissa,A.Dorra,and K. M. Metwally, “Flowcytometric analysis of T-lymphocytes and serum tumour necrosis factor alpha (TNF-alpha) levels in Schistosoma mansoni patients,” Journal of the Egyptian Society of Parasitology, vol. 37, no. 3, pp. 1065–1074, 2007. [46] M. O. Li, Y. Y. Wan, S. Sanjabi, A. K. L. Robertson, and R. A. Flavell, “Transforming growth factor-β regulation of immune responses,” Annual Review of Immunology, vol. 24, pp. 99–146, 2006. [47] J. Massague, J. Andres, L. Attisano et al., “TGF-β receptors,” Molecular Reproduction and Development,vol.32,no.2,pp. 99–104, 1992. [48] R. R. El-Gamal, S. M. Nada, N. E. Moustafa et al., “Relation- ship between serum cytokines profiles and hepatic fibrosis in schistosomiasis mansoni: an experimental study,” Journal of the Egyptian Society of Parasitology, vol. 39, no. 3, pp. 907–916, 2009. [49] M. L. Burke, M. K. Jones, G. N. Gobert, Y. S. Li, M. K. Ellis, and D. P. McManus, “Immunopathogenesis of human schistosomiasis,” Parasite Immunology, vol. 31, no. 4, pp. 163–176, 2009. Hindawi Publishing Corporation Journal of Parasitology Research Volume 2012, Article ID 849132, 11 pages doi:10.1155/2012/849132

Review Article New Frontiers in Schistosoma Genomics and Transcriptomics

Laila A. Nahum,1, 2, 3 Marina M. Mourao,˜ 1 and Guilherme Oliveira1, 2

1 Genomics and Computational Biology Group, Centro de Pesquisas René Rachou (CPqRR), Instituto Nacional de Ciência e Tecnologia em Doenças Tropicais, Fundaçao˜ Oswaldo Cruz (FIOCRUZ), 30190-002 Belo Horizonte, MG, Brazil 2 Centro de Excelência em Bioinformatica´ (CEBio), Fundaçao˜ Oswaldo Cruz (FIOCRUZ), 30190-110 Belo Horizonte, MG, Brazil 3 Faculdade Inforium´ de Tecnologia, 30130-180 Belo Horizonte, MG, Brazil

Correspondence should be addressed to Guilherme Oliveira, oliveira@cpqrr.ﬁocruz.br

Received 10 August 2012; Accepted 16 October 2012

Academic Editor: Sergio Costa Oliveira

Copyright © 2012 Laila A. Nahum et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Schistosomes are digenean blood flukes of aves and mammals comprising 23 species. Some species are causative agents of human schistosomiasis, the second major neglected disease affecting over 230 million people worldwide. Modern technologies including the sequencing and characterization of nucleic acids and proteins have allowed large-scale analyses of parasites and hosts, opening new frontiers in biological research with potential biomedical and biotechnological applications. Nuclear genomes of the three most socioeconomically important species (S. haematobium, S. japonicum,andS. mansoni) have been sequenced and are under intense investigation. Mitochondrial genomes of six Schistosoma species have also been completely sequenced and analysed from an evolutionary perspective. Furthermore, DNA barcoding of mitochondrial sequences is used for biodiversity assessment of schistosomes. Despite the efforts in the characterization of Schistosoma genomes and transcriptomes, many questions regarding the biology and evolution of this important taxon remain unanswered. This paper aims to discuss some advances in the schistosome research with emphasis on genomics and transcriptomics. It also aims to discuss the main challenges of the current research and to point out some future directions in schistosome studies.

1. Introduction more commonly found in cattle, is believed to form hybrids between S. mattheei and S. haematobium thus increasing its Schistosomatidae (Platyhelminthes: Digenea) includes sev- snail and definitive host range [3]. eral digenetic endoparasites with complex life cycles, whose Schistosomiasis, a chronic and debilitating disease, is developmental stages alternate between intermediate (fresh- considered by the World Health Organization as one of water gastropods) and definitive hosts (birds, reptiles, fishes, the most serious public health issues and the second most and mammals) [1]. These parasites differ from other blood prevalent tropical disease with high morbidity in the world flukes in having separate sexes. Another important feature [5]. Schistosomiasis is endemic in 77 countries [6] and its is the increased longevity (over 5 years) of the Schistosoma transmission is dependent on the existence and distribution species in the human host [1]. of intermediate hosts. It is estimated that 237 million people Schistosoma, the avian and mammalian blood flukes, require treatment worldwide and that 600 to 779 million is the best studied genus of Schistosomatidae [1]. Several people live in endemic areas, under infection risk [6]. Yet, species are described, six of which infect humans causing exacerbating this scenario, this disease is responsible for the schistosomiasis: S. haematobium, S. intercalatum, S. japon- loss of 1.7 to 4.5 millions of years of life in the world, icum, S. malayensis, S. mansoni,andS. mekongi. Other measured by disability-adjusted life years (DALY) [7], one of species are known to infect a broad range of mammals the highest indexes among all the neglected tropical diseases. such as hippopotamus, rodents, carnivores, and ruminant Control of schistosomiasis represents a great challenge and animals like buffalo, cattle, goat, lechwe, and sheep. Some it is based on drug treatment (Praziquantel), snail control, hybrid species are reported [1–4]. For example, S. mattheei, improved sanitation, and health education [5]. 2 Journal of Parasitology Research

The development of powerful and scalable methods identification of genetic loci that determine important traits to analyse nucleic acids and proteins has changed the such as host specificity, virulence, and drug resistance. way biological data is surveyed. The application of such In fact, although the quality and annotation of the S. technologies, together with the development of powerful mansoni genome sequence data have been greatly improved computational tools and methods, have expanded our (see below); the corresponding data of S. haematobium perspective of schistosome biology and allowed a better and S. japonicum are still considered drafts [16–19]. Their understanding of processes such as host-parasite interaction genome sequences are distributed in a large number of [8–10]. contigs; therefore extensive work is required. Constant data This paper aims to discuss some advances in schistosome refinement is necessary to provide a reliable comprehension research with emphasis on genomics and transcriptomics. of the genome and gene models and to provide a curated First, we summarize the current status of sequencing projects annotation. of nuclear and mitochondrial genomes. We also discuss The S. mansoni genome annotation has been improved some aspects of evolutionary genomics and biodiversity of by combining different sequencing strategies [19]. Such schistosomes. Then, we present key findings in transcrip- strategies include new assembly of existing capillary reads tomic analyses. Finally, we point out the main challenges of supplemented with an additional ∼90,000 fosmid and BAC the current research and suggest some future directions in end sequences, deep sequencing of clonal DNA (NMRI Schistosoma genomic and transcriptomic studies. strain, Puerto Rican origin) using 108-based paired reads on Illumina Genome Analyzer IIx platform, and RNA-seq data of different parasite life stages. 2. Schistosoma Genomics Progress on the knowledge of S. mansoni genome features is still expanding, especially with the application of the next- In order to strengthen traditional methods and provide generation sequencing platforms and single nucleotide poly- new information to improve success towards schistosomiasis morphism typing methods. For instance, 14 new microsatel- control, the scientific community joined efforts to assess lite markers were recently identified by de novo assembly the Schistosoma genomic information, starting in 1994, as of massive sequencing reads; these new features can be an initiative of WHO/TDR (UNICEF-UNDP-World Bank- employed for pedigree studies [22]. Yet, newer sequencing WHO Special Programme for Research and Training in Tropical Diseases) [11, 12]. At that time, only a few hundred technologies will allow for the development of population expressed sequence tags (ESTs) from S. mansoni were avail- genomics studies of field, laboratory, and clinical isolates. able [13]. The WHO/TDR support opened the possibility As a result of the availability of the Schistosoma genome ff to generate data that could be translated into new tools for sequence data, comparative analyses across di erent species ff schistosomiasis diagnostics and treatment, representing the became possible bringing together di erent areas of research “kickoff”ofSchistosoma nuclear genome studies. [21, 23, 24]. Comparative genomics of three Schistosoma species revealed several conserved features such as the overall GC content, sequence identity, presence of repetitive 2.1. Nuclear Genomes. The karyotype of known Schistosoma elements, and synteny [18]. This genome-wide analysis species comprises seven pairs of autosomes and one pair of corroborates previous genetic studies and supports the = = sex chromosomes (female ZW, male ZZ), ranging in size evolutionary hypothesis of S. haematobium and S. mansoni from 18 to 73 megabases (Mb) [14, 15]. as the most related species, followed by S. japonicum, to the The nuclear genome of three Schistosoma species was exclusion of other Trematoda such as Clonorchis, Fasciola, sequenced (Table 1). The S. japonicum (Anhui isolate) and and Schmidtea. S. mansoni (PuertoRicoisolate)genomesweredecodedand simultaneously published as an initiative of the Wellcome Trust Sanger Center Institute in collaboration with the 2.2. Mitochondrial Genomes. Mitochondrial genes have been Institute for Genomic Research (TIGR) and the Schistosoma used as molecular markers for species and strain identifica- japonicum Genome Sequencing and Functional Analysis tion, which is key to a variety of studies such as phylogenetics, Consortium [16, 17]. Recently, the S. haematobium (Egyp- population genetics, biogeography, and molecular ecology tian isolate) genome was sequenced opening new possibilities [25–29]. Besides helping to elucidate the evolutionary rela- in comparative genomics of schistosomes [18]. tionships among taxa, mitochondrial genes have been suc- S. haematobium, S. japonicum,andS. mansoni carry a cessfully applied into epidemiological studies, monitoring, nuclear genome of 385, 397, and 363 Mb composed by and control of microbes, parasites, and vectors of medical approximately, 13,073, 13,469, and 10,852 protein-coding and socioeconomic importance. The analysis of the whole genes, respectively [16–19]. It is worth to mention that the mitochondrial genome of diverse taxa has changed the number of predicted protein-coding genes does not reflect perspective of such studies. the genome structure per se, but the actual state of analyses The mitochondrial genome of six Schistosoma species of the different draft genome data. was sequenced (Table 1) including S. haematobium The genetic linkage map of S. mansoni was obtained (NC 008074), S. japonicum (NC 002544), S. mansoni and allowed the refinement of the genome sequencing data (NC 002545), S. malayensis, S. mekongi (NC 002529), providing a means for gene discovery and gene function and S. spindale (NC 008067) [27, 30–32]. These genomes analysis [20, 21]. More importantly, it has allowed the range in size from 13,503 to 16,901 bp and encode 36 genes Journal of Parasitology Research 3

Table 1: Availability of genomic and transcriptomic data for Schistosoma species.

Taxid Taxon ncDNA mtDNA ESTs Barcoding 6184 Schistosoma bovis No No No Yes 6186 Schistosoma curassoni No No No Yes 230327 Schistosoma edwardiense No No No Yes 393876 Schistosoma guineensis No No No Yes 6185 Schistosoma haematobium Yes Yes Yes Yes 157462 Schistosoma hippopotami No No No Yes 198245 Schistosoma incognitum No No No Yes 216970 Schistosoma indicum No No No Yes 6187 Schistosoma intercalatum No No No Yes 6182 Schistosoma japonicum Yes Yes Yes Yes 646316 Schistosoma kisumuensis (∗)NoNoNoYes 216972 Schistosoma leiperi No No No Yes 53353 Schistosoma malayensis No Yes No Yes 6183 Schistosoma mansoni Yes Yes Yes Yes 48269 Schistosoma margrebowiei No No No Yes 31246 Schistosoma mattheei No No No Yes 38744 Schistosoma mekongi No Yes No Yes 216971 Schistosoma nasale No No No Yes 6188 Schistosoma rodhaini No No No Yes 191505 Schistosoma sinensium No No No Yes 230328 Schistosoma sp. Uganda-JATM-2003 No No No Yes 6189 Schistosoma spindale No Yes No Yes Taxid: NCBI taxonomy identifier. ncDNA: nuclear DNA genome (SchistoDB.org). mtDNA: mitochondrial DNA genome (NCBI Organelle Genome Resources). ESTs: data from NCBI dbEST (release 120701, July 1, 2012). Barcoding: DNA barcoding used on cox1 sequences (BOLD Systems). ∗Schistosoma sp. BH-2009. comprising two ribosomal genes (large and small subunit evolutionary history of Schistosoma especially due to the rRNA genes), and 22 transfer RNA (tRNA) genes, as well as absence of fossil records. Molecular markers have been used 12 protein-encoding genes (atp6, cob, cox1–3, nad1–6, and to test hypotheses in a variety of Schistosoma phylogenetic, nad4L). The atp8 gene, commonly found in other phyla, is phylogeographic and epidemiological studies [4, 25, 34–36]. absent from Platyhelminthes, whose mitochondrial genomes Traditionally, these markers include the nuclear ribosomal have been analysed [31]. Moreover, each Schistosoma RNA genes (18S, 5.8S, and 28S) and the internally tran- mitochondrial genome contains a long noncoding region scribed spacer (ITS) region besides a number of mitochon- that is divided into two parts by one or more tRNA genes, drial genes (cob, cox1–3, nad1–6, etc.). which is found in other Platyhelminthes and vary in length Based on mitochondrial data from different studies, a according to each taxon. standard phylogeny of 23 Schistosoma species was proposed Comparative mitogenomics have revealed a highly con- [25]. Six clades were identified that correlate with the served gene order among distinct Platyhelminthes with geographic distribution of the species analysed. These clades the exception of some Schistosoma species [25, 30–33]. include (1) the S. japonicum complex (S. japonicum, S. The mitochondrial genome of the Asian schistosomes, S. malayensis, S. mekongi, S. ovuncatum,andS. sinensium) japonicum and S. mekongi, displays the same gene order as found in Central and South Eastern Asia; (2) the S. other Digenea and Cestoda [25, 32]. hippopotami clade (S. edwardiense and S. hippopotami) In contrast, the gene order in the mitochondrial genome distributed in Africa; (3) the proto-S. mansoni clade (S. of S. haematobium, S. mansoni,andS. spindale is strik- incognitum and S. turkestanicum) in Central Asia, Near East, ingly different from other taxa [25, 30]. The unique gene and Eastern Europe; (4) the S. mansoni clade (S. mansoni and order rearrangements identified in these species constitute S. rodhaini) found in Africa and South America; (5) the S. valuable information to the reconstruction of the phyloge- haematobium clade (S. bovis, S. curassoni, S. guineensis, S. netic relationships of Schistosoma and other Platyhelminthes haematobium, S. intercalatum, S. kisumuensis, S. leiperi, S. (Section 3.1). margrebowiei,andS. mattheei) occurring in different parts of Africa; (6) the S. indicum clade (S. indicum, S. nasale,and 3. Evolutionary Genomics and Biodiversity S. spindale) found in India and South Asia. There are divergent opinions concerning the origin of the 3.1. Evolutionary Genomics of Schistosoma. The use of genus Schistosoma and its intermediate snail host [25, 37– genomic data is valuable to the understanding of the 39]. The “out of Asia” hypothesis is the most generally 4 Journal of Parasitology Research accepted, indicating a migration followed by dispersion of This study also demonstrated that SmNR4A expression was these parasites from Asia to Africa [25]. In this regard, studies regulated throughout parasite development. of molecular phylogeny suggested that the genus Schistosoma Thioredoxin and glutathione systems, involved in a had its origin in Asia and (at least two descendants) colonized variety of cellular processes including antioxidant defense, the African continent independently, where they easily differ in parasitic and free-living Platyhelminthes [47]. By radiated, becoming exclusive parasites of planorbid snails. applying different phylogenetic methods and experimental Returning to Asia, the parasites diversified into groups of testing, it has been shown that the canonical enzymes of such species, characterized by the position of the spike in the eggs systems were lost in the parasitic lineages. [38]. The molecular phylogeny of seven Schistosoma species suggested that schistosomes were endoparasites of rodents and ruminants and were transmitted to the first hominids 3.2. DNA Barcoding for Biodiversity Assessment. The avail- in Africa as a migration to savanna areas occurred [40]. ability of sequencing data from mitochondrial genomes from However,inordertobetterdefinetheSchistosoma origin, closely related taxa to Schistosoma has provided a basis for there is a need to sample a broader range of species. studies ranging from phylogenetic systematics to molecular Most of the widely accepted Schistosoma phylogenies are ecology and so forth [27, 29, 48, 49]. Importantly, it allowed primarily based on sequence alignments of mitochondrial the assessment of molecular markers prior to broad scale genes such as cox1–3, and nad1–6, [25, 30]. Other studies sampling by comparing data from individual genes versus use changes in the mitochondrial gene order as phylogenetic completely sequenced mitochondrial genomes. markers. Indeed, these gene rearrangements, considered Data from African and Asian schistosomes were com- to be rare evolutionary events, have been applied to an pared to an S. mansoni intraspecific dataset showing a increasing number of studies (cf. [41]). positive correlation between polymorphism and species As mentioned before, major changes in the mitochon- divergence [27]. A positive correlation rather than random drial gene order were identified in some Schistosoma species distribution was identified for all mitochondrial genes with (Section 2.2), more specifically in the African clades (S. the exception of cox1 and nad1. Furthermore, partial mansoni and S. haematobium) and the S. indicum group sequences of cox1 have been shown not to be the ideal (S. spindale). This provides further evidence for the Asian marker for either species identification or population studies ancestry of schistosomes and corroborates the concept of the of Schistosoma species. Instead, authors have suggested the reinvasion of Asia by members of the S. indicum group from use of cox3 and nad5 sequences for both phylogenetic and Africa [27, 30]. population studies of such species [27]. Besides reconstructing the phylogenetic relationships of DNA barcoding was performed for samples of S. haema- diverse taxa, the evolutionary framework has been applied to tobium using cox1 and nad1 as molecular markers [49]. improve functional annotation of genes and gene products, The results supported the identification of group 1 and 2 as well as to study gene/protein family evolution [41]. haplotypes based on phylogenetic analysis. Moreover, no Phylogenomics has allowed the identification and character- change in genetic diversity was detected across samples ization of all eukaryotic protein kinases encoded in the S. collected over different time points. This approach allowed mansoni genome and improved the functional annotation the development of new assays based on group-specific PCR of over 40% of them, highlighting the molecular diversity of primers and SNaPshot probes to assist genetic screening of these enzymes [42, 43]. schistosome isolates. A phylogenetic analysis was also employed in the clas- Another study employing mitochondrial sequences was sification of S. mansoni histone proteins, which play a performed in Schistosoma cercariae harvested from Biom- key role in epigenetic modifications that might reflect the phalaria choanomphala as part of parasitological and mala- parasite complex lifestyle [44]. By applying an evolutionary cological surveys for S. mansoni across Lake Victoria [48]. framework to analyse a large sequence dataset from a broad DNA barcoding of cercarial samples revealed the presence range of organisms, functional annotation of the S. mansoni of hybrid species between S. mansoni and S. rodhaini, histone proteins was improved. which is typically found in small mammals. Moreover, the Moreover, phylogenomics has unraveled the distinct phylogenetic analysis identified a new sublineage within S. evolutionary histories of three expanded endopeptidase rodhaini. families in S. mansoni [45]. This analysis included members DNA barcoding studies are underway for an increasing number of Schistosoma species (Table 1), whose data of the metallopeptidase M8, serine peptidase S1, and aspartic is made available through the Barcode of Life Database peptidase A1 families, which were originated from successive (BOLD) system (http://www.boldsystems.org/). This initia- events of gene duplication followed by divergence in the par- tive includes contributors from the Biodiversity Institute of asite lineage after its diversification from other metazoans. Ontario, Canada, and the University of New Mexico, USA, Other protein families such as nuclear receptors and among others. Other Schistosomatidae genera are subject antioxidants have been characterized in S. mansoni by to DNA barcoding analysis such as Allobilharzia, Austro- applying evolutionary and functional approaches [46, 47]. bilharzia, Bilharziella, Bivitellobilharzia, Dendritobilharzia, The identification of SmNR4A, a member of the nuclear Gigantobilharzia, Griphobilharzia, Heterobilharzia, Macrobil- receptor subfamily 4, and its relatedness to the human harzia, Orientobilharzia, Ornithobilharzia, Schistosomatium, homologs were corroborated by phylogenetic analysis [46]. and Trichobilharzia. Journal of Parasitology Research 5

Advances in DNA barcoding of a broader range of by parasite pairing. In addition, gender biased alternative Schistosomatidae will create a framework for species iden- splicing patterns were observed, which are perhaps involved tification from environmental and clinical samples as well in the generation and maintenance of the sexual dimorphism as specimens deposited in biological collections. Moreover, [57]. DNA barcoding analysis will help reconstructing the evo- Later, studies profiled the S. mansoni gene expression lutionary relationships across different Platyhelminthes and in different life stages broadening our understanding of ultimately improve our understanding of parasite biology factors controlling schistosome development [8, 53, 58]. and evolution. While comparing different parasite life stages, information acquired from microarray datasets highlighted putative antischistosome candidate molecules to be used as vaccine 4. Schistosoma Transcriptomics targets including Dyp-type peroxidases, fucosyltransferases, Transcriptomic analyses are valuable not only for studies of G-protein coupled receptors, leishmanolysins, tetraspanins, differential regulation in gene expression, but also for the and the netrin/netrin receptor complex [59]. identification of splicing variants. These approaches have Some of these candidates (fucosyltransferases, leishman- proved to be essential in the identification of gene-coding olysins, and tetraspanins) are members of protein families, regions during the process of genome annotation. which are expanded in the S. mansoni predicted proteome In this section, we aim to review the achievements in comparison with other metazoans as inferred by phyloge- in Schistosoma transcriptomic studies that range from the nomic analysis (http://phylomedb.org/). Leishmanolysins, whole organism and tissue analyses to those performed at which are members of the metallopeptidase M8 family, the cellular level. Transcriptomics in physiological conditions were analysed in detail corroborating the potential of these and in response to a variety of treatments are investigated enzymes as future therapeutic targets [45]. with respect to the interaction with the host as well as other Another study identified upregulated genes during the environments. This section summarizes the accomplish- first five days of schistosomula development in vitro that ments of gene function studies and the diverse techniques are involved in blood feeding, tegument and cytoskeletal that are being employed. development, cell adhesion, and stress responses [8]. The highly upregulated genes included a tegument tetraspanin Sm-tsp-3, a protein kinase, a novel serine protease and 4.1. Schistosoma Transcriptomics. A fine tuning in gene ex- serine protease inhibitor, and intestinal proteases belonging pression of parasites must occur to enable them to survive to distinct mechanistic classes. Other groups have analysed in such a complex and extremely diverse milieu, adapting immunity towards Schistosoma using protein microarrays to diverse environments as evading the immune system and, as a vaccine discovery tool. They selected a subset of ff at the same time, exploiting di erent hosts. Transcriptomic proteins from genomic, transcriptomic, and proteomic data studies came along allowing a temporal and quantitative to perform experimental validation aiming at identifying analyses of gene expression, which are crucial to completely novel antischistosome vaccine candidates [60, 61]. ff exploit di erent datasets and assess parasite biology at the Recently, an elegant approach to investigate specific molecular level. tissuessuchasgastrodermisandbothgendersreproduc- ff Aiming to elucidate the di erential regulation on the tive tissues was performed by applying laser microdissec- Schistosoma transcriptomics, a large number of projects tion microscopy combined to microarray. These studies employed diverse strategies, such as the ones using expressed brought the perspective of expediting tissue-specific profiling sequence tags (ESTs) [50], open reading frame expressed [62, 63]. sequence tags (ORESTEs) [51], and serial analysis of gene Additionally, expression profiles of S. japonicum were expression (SAGE) [52, 53]. explored in studies comparing two Chinese isolates (Anhui The increasing number of projects resulted in the pro- and Zhejiang), as well as the Anhui Chinese and Philippines duction of a vast amount of sequence data and their isolates [55, 64]. Later, an interspecies study compared the availability in public databases. It is noteworthy that only S. japonicum and S. mansoni transcriptomes. Surprisingly, S. japonicum, S. mansoni and, not until very recently, S. despite their large phenotypic differences, there was a haematobium had some of their transcriptome explored reduced number of differentially expressed genes based on (Table 1). the expression profile, which might reflect a limitation of the technique (preferential hybridization related to species 4.2. Exploring the Transcriptome. The large number of tran- polymorphisms) [8]. Bypassing these pitfalls, the next gener- scriptome projects accelerated the studies in this area by ation sequencing platforms allow exploiting expression data providing means to the use of complementary approaches, (RNA-seq) of any organism without previous knowledge of especially microarray analysis. Together, these approaches the genome or transcriptome and, further, will reveal new have been used to answer a wide range of questions. In the features of the parasite transcriptional landscape. early years, microarray datasets conceived the profile of gene The most recent achievement in this area was the expression between genders of Schistosoma species such as refinement of the genomics and transcriptomics data using a S. japonicum and S. mansoni [54–56]. This greatly extended combination of Sanger capillary sequencing, next generation the number of known sex-associated genes and also provided DNA sequencing, genetic markers, and RNA-seq data from new insights into changes in gene expression promoted several S. mansoni life stages [19]. A previous study, explored 6 Journal of Parasitology Research the transcriptome of S. mansoni adult males obtaining 11 and labor-demand functional studies, the difficulties to novel microexon genes (MEGs), as well as mapping 989 and nurture the complete life cycle in vitro, and the absence of 1196 contigs to intergenic and intronic genomic regions, immortalized cell lines, which has delayed the development respectively. This data could represent new protein-coding of transgenesis tools and models [71]. genes or noncoding RNAs (ncRNAs) [65]. Lately, the posttranscriptional suppression of genes Together, these approaches have significantly advanced through RNA interference (RNAi) techniques has been a our understanding of the dynamics parasite transcriptomes promise to fetch progress on new interventions for schis- [9, 65]. However, there is a need to polish and translate tosomiasis and other helminthiases. In 2003, two pioneer sequence data and gene expression profiles into functional investigations marked the use of this technique in S. information by computational predictions and experimental mansoni research. Simultaneously gene knockdown of glyc- characterization. The vast amount of available data opened eraldehyde-3-phosphate dehydrogenase (GAPDH) and a a myriad of possibilities towards the elucidation of gene glucose transporter (SGTP1) in the larval stage (sporocysts) functions. It is time for data integration. [72] and cathepsin B in adult worms [73]ofS. mansoni were reported. Currently, this powerful technique is widely used in 4.3. Posttranscriptional Mechanisms. Transcriptomics is also schistosomes and has been employed in two studies of func- a key approach to study posttranscriptional regulatory tional screening. The first study encompassed the exposure mechanisms such as alternative and transsplicing [57, 66– of sporocysts by soaking parasites to dsRNA targeting 33 68]. It is known that schistosomes use such mechanisms to genes, from calcium binding proteins, transcription factors, keep their intricate life cycle. to receptors, and antioxidant enzymes [74]. In this work, Alternative splicing of transcripts of secreted proteins, despite the efficacy of this tool to deliver suppression of 11 like MEGs and polymorphic mucin isoforms, has been different genes, it showed that, in S. mansoni, there is a gene- largely studied in schistosomes [51, 66]. This posttranscrip- to-gene variability and exposure to dsRNA could trigger tional processing has been proposed as a distinct genetic gene specific upregulation. Later, in a different study using system to generate protein variation that would allow electroporation to expose schistosomula to dsRNA of 11 parasite adaptation to the immune response of different target genes (expressed in distinct tissues), the efficiency of hosts. Additionally, alternative splicing has been shown to RNAi in a target specific fashion was observed [75]. Other generate protein variation involved in transcriptional control issues with the use of RNAi have been widely discussed, not and splicing [57]. In the case of SmCA150, a spliceosome only in schistosomes, but also in other helminth parasites, interacting transcriptional cofactor, it could impact many showing that care should be taken when conducting and other transcripts leading to several effects in the parasite, evaluating such experiments [76]. especially in sexual dimorphism. Despite the difficulties in exploring gene function by Spliced leader (SL) transsplicing has been shown to be RNAi in schistosomes, many interesting advances have been an important posttranscriptional regulation mechanism for achieved. Many proteases (cathepsins and aminopeptidases) S. mansoni [69]. At least 11% of all transcripts has been [77–79], kinases [80], multidrug resistance transporters [81], described as undergoing transsplicing in this organism [19]. and type V collagen [82], among other proteins have been SL-RNAs are small ncRNAs, products of small intronless studied in diverse life stages of S. japonicum and S. mansoni. genes, transcribed by DNA polymerase II and tandemly Recently, this tool has been described targeting repeated in the genome [67]. tetraspanin 2 in diverse developmental stages of S. Differently from nematodes, S. mansoni carries a single haematobium indicating the RNAi feasibility in this species SL sequence, which is composed by 36 nucleotides with an [83]. Tetraspanins belong to a family of integral membrane 3-terminal AUG codon. Although the role of transsplicing proteins present in the outer surface membranes of the is poorly understood, there is an evidence suggesting that SL parasite tegument. Studies have shown that tetraspanins play transsplicing would function providing an initiator methio- important roles in the tegument development, maturation, nine for translation initiation of some recipient mRNAs in or stability [84]. Platyhelminthes [68]. This mechanism is also involved in Importantly, the knockdown of peroxiredoxin [85] the resolution of polycistronic operons in monocistronic and thioredoxin glutathione reductase [86], antioxidant transcripts in S. mansoni [19, 70]. enzymes, were amongst the first reports of lethality after gene silencing in the parasite, demonstrating the potential 4.4. Transcriptome Data and Gene Function. Despite the of RNAi on drug target discovery. Moreover, enzymes such great potential revealed by the increasing number of genome as glutathione-S-transferases (GST26 and GST28), peroxire- and transcriptome studies in schistosomes, there are still doxins (Prx1 and Prx2), and superoxide dismutase have been several Schistosoma species that remain neglected. In contrast shown to play important roles in the protection of S. mansoni to the vast amount of data available in public databases, sporocysts in the host-parasite interplay. Knockdown of limited data has been decoded in functional studies and/or those targets increased parasite susceptibility to oxidative in the development of successful tools to fight schistosomes. stress and to intermediate host immune cells as described This slow evolving scenery might be due to several reasons. elsewhere [87]. Some of them include the convolution of schistosomes Recently, significant progress has been made in the area life cycle, the complex interaction with their hosts, time of schistosome transgenesis. Researchers are developing new Journal of Parasitology Research 7 tools for the introduction and expression of homologous 6. Conclusions and Future Directions or heterologous gene constructs via lentiviruses, replication- defective retroviruses, transposons, retrotransposons, and The -omics studies came along with the aim to fill the gaps DNA plasmid vectors [9]. Sophisticated systems applied to of long-established approaches, enabling the use of a broad assess gene function in the parasite are being achieved [88– range of experimental and computational methods and 91]. To date, eggs and miracidia are indicated as the preferred contributing to dissect the biological basis of host-parasite life stages for successful transformation approaches in order interaction, diagnostics improvement, and the discovery of to enter the germline [92]. new drug and vaccine targets. In summary, results from such independent studies Nuclear and mitochondrial genomes of some Schisto- have shown that schistosome transgenesis works in all the soma species were completely sequenced opening new fron- developmental stages tested and that some success has been tiers for comparative analyses, which will improve our under- achieved in integrating genes into the germline [88–92]. standing of parasite biology and evolution. Other genomes to Thus, genetic knockdown experiments in schistosomes are be analysed include Schistosoma species from each of the six possible. clades currently recognized, and other representatives of the Schistosomatidae. Improving accuracy/quality of genome assembly and annotation will be crucial for such analyses. ff 5. Genomic and Transcriptomic Databases Despite the e orts in the characterization of genomes and transcriptomes of distinct Schistosoma species, many Altogether, the increasing information on schistosome questions regarding the biology and evolution of this impor- ffi genomes and transcriptomes is now raising the possibilities tant taxon remain unanswered. These di culties might be to better explore molecular techniques in the field of due to the size of schistosome genomes (∼10-times larger parasitology, genomics, and computational biology allowing compared to protozoan parasites), the complexity of its life the development of approaches to unravel mechanisms of cycle, the presence of rare features in posttranscriptional parasite infection, host-parasite interaction, and so forth regulation (e.g., transsplicing), and little integration of the [21, 23, 24]. current information available, among other reasons. In order to accelerate both parasite research and devel- There is an urgent need for data integration to help inter- opment of effective tools for schistosomiasis diagnostics and preting the massive amount of data generated over the years treatment, user-friendly databases have been created and in this and other areas or research. The need for biocuration made publicly available to the scientific community [9, 93– is essential to guide this process. We envision that, in the 95]. Here, we highlight some of these major resources. future, more transcriptomic and proteomic data will be used SchistoDB integrates genome and proteome sequence to improve the understanding of the origin and evolution of data along with functional annotation of genes and gene Schistosomatidae. The analysis of structural data generated products of S. haematobium, S. japonicum,andS. mansoni by experimental or computational approaches will also shed [93]. This resource also covers results from large-scale light in the biodiversity of schistosomes at the molecular analysis including ESTs, metabolic pathways, and candidate level. drug targets. In conclusion, the availability of schistosome genome HelmCoP (Helminth Control and Prevention) integrates and transcriptome data has provided an unprecedented functional, structural, and comparative genomics data of resource for many research areas. Limitations do exist and helminths from different taxonomic groups and distinct should be addressed to allow advances in understanding hosts (plant, animal, and human) [94]. HelmCoP offers a schistosome biology and evolution and ultimately support comprehensive suite of structural and functional annotations vaccine and drug development against schistosomiasis. to support comparative analyses and host-parasite interaction studies. List of Abbreviations GeneDB is an annotation database that brings together data from a broad range of pathogens and closely related atp6: ATP synthase F0 subunit6 organisms [95]. GeneDB offers database-driven annotation atp8: ATP synthase F0 subunit 8 tools and pipelines and includes manually curated informa- bp: Base pairs tion maintained by computational and biological scientists. cob: Apocytochrome b Collectively, these computational resources support bio- cox1: Cytochrome c oxidase subunit 1 logical and biomedical research and open new frontiers cox1–3: Cytochrome c oxidase subunit 1 to 3 in evolutionary and functional genomics, transcriptomics, DALY: Disability-adjusted life years and proteomics of Schistosoma species and other parasites. dbEST: Database of expressed sequence tags Besides biodiversity assessment at the molecular level, these dsRNA: Double strand DNA resources provide a framework for the identification and ESTs: Expressed sequence tags prioritization of vaccine and drug targets against human MEGs: Microexon genes diseases. As the scientific community progresses in data nad1–6: NADH dehydrogenase subunit 1 to 6 integration and interpretation, we will meet the goals of nad4L: NADH dehydrogenase subunit 4L current and future initiatives to improve human health [9, ncRNAs: Noncoding RNAs 12, 23, 24]. ORESTEs: Open reading frame expressed sequence tags 8 Journal of Parasitology Research

RNAi: RNA interference biology and anthelmintic discovery,” Trends in Parasitology, RNA-seq: RNA sequencing vol. 27, no. 12, pp. 555–564, 2011. SAGE: Serial analysis of gene expression [11] G. Oliveira, G. Franco, and S. Verjovski-Almeida, “The Bra- SL: Spliced leader zilian contribution to the study of the Schistosoma mansoni TDR: Special Programme for Research and Training transcriptome,” Acta Tropica, vol. 108, no. 2-3, pp. 179–182, in Tropical Diseases 2008. [12]P.T.LoVerde,H.Hirai,J.M.Merrick,N.H.Lee,andN. UNDP: United Nations Development Programme El-Sayed, “Schistosoma mansoni genome project: an update,” UNICEF: United Nations Children’s Fund Parasitology International, vol. 53, no. 2, pp. 183–192, 2004. WHO: World Health Organization [13]G.R.Franco,M.D.Adams,M.B.Soares,A.J.G.Simpson,J. C. Venter, and S. D. J. Pena, “Identification of new Schistosoma mansoni genes by the EST strategy using a directional cDNA Acknowledgments library,” Gene, vol. 152, no. 2, pp. 141–147, 1995. The research leading to these results has received fund- [14] A. J. G. Simpson, A. Sher, and T. F. McCutchan, “The genome of Schistosoma mansoni: isolation of DNA, its size, bases and ing from the European Community’s Seventh Frame- repetitive sequences,” Molecular and Biochemical Parasitology, work Programme (FP7/2007–2013 FP7/2007–2011) under vol. 6, no. 2, pp. 125–137, 1982. Grant agreement number 241865. This work was also [15] A. I. Grossman, R. B. Short, and G. D. Cain, “Karyotype partially supported by the National Institutes of Health- evolution and sex chromosome differentiation in schistosomes NIH/Fogarty International Center (TW007012 to GO), the (trematoda, schistosomatidae),” Chromosoma, vol. 84, no. 3, National Council for Research and Development, CNPq pp. 413–430, 1981. (CNPq Research Fellowship 306879/2009-3 to GO, INCT- [16]M.Berriman,B.J.Haas,P.T.Loverdeetal.,“Thegenome DT 573839/2008-5 to GO, CNPq-Universal 476036/2010-0 of the blood fluke Schistosoma mansoni,” Nature, vol. 460, no. to LAN, and 480576/2010-6 to MMM), and the Research 7253, pp. 352–358, 2009. Foundation of the State of Minas Gerais (FAPEMIG) (CBB- [17] Y. Zhou, H. Zheng, Y. Chen et al., “The Schistosoma japonicum 1181/08 and PPM-00439-10 to GO and CBB-APQ-01715/11 genome reveals features of host-parasite interplay,” Nature, vol. 460, no. 7253, pp. 345–351, 2009. to MMM). The authors are very grateful to Larissa Lopes [18] N. D. Young, A. R. Jex, B. Li et al., “Whole-genome sequence Silva and two anonymous reviewers for their constructive of Schistosoma haematobium,” Nature Genetics, vol. 44, no. 2, feedback and valuable suggestions, which greatly improved pp. 221–225, 2012. this paper. [19] A. V. Protasio, I. J. Tsai, A. Babbage et al., “A systematically improved high quality genome and transcriptome of the human blood fluke Schistosoma mansoni,” PLoS Neglected References Tropical Diseases, vol. 6, no. 1, Article ID e1455, 2012. [20] C. D. Criscione, C. L. L. Valentim, H. Hirai, P. T. LoVerde, [1] B. Gryseels, “Schistosomiasis,” Infectious Disease Clinics of and T. J. C. Anderson, “Genomic linkage map of the human North America, vol. 26, no. 2, pp. 383–397, 2012. blood fluke Schistosoma mansoni,” Genome Biology, vol. 10, no. [2] T. Huyse, B. L. Webster, S. Geldof et al., “Bidirectional in- 6, article R71, 2009. trogressive hybridization between a cattle and human schis- [21] A. Tait, “Genetics and genomics converge on the human blood tosome species,” PLoS Pathogens, vol. 5, no. 9, Article ID fluke,” Genome Biology, vol. 10, no. 6, article 225, 2009. e1000571, 2009. [22] J. M. J. Lepesant, D. Roquis, R. Emans et al., “Combination [3] F. J. Kruger and A. C. Evans, “Do all human urinary infections of de novo assembly of massive sequencing reads with with Schistosoma mattheei represent hybridization between S. classical repeat prediction improves identification of repetitive haematobium and S. mattheei?” Journal of Helminthology, vol. sequences in Schistosoma mansoni,” Experimental Parasitology, 64, no. 4, pp. 330–332, 1990. vol. 130, no. 4, pp. 470–474, 2012. [4] M. L. Steinauer, M. S. Blouin, and C. D. Criscione, “Applying [23]R.A.Wilson,P.D.Ashton,S.Braschi,G.P.Dillon,M. evolutionary genetics to schistosome epidemiology,” Infection, Berriman, and A. Ivens, “Oming in on schistosomes: prospects Genetics and Evolution, vol. 10, no. 4, pp. 433–443, 2010. and limitations for post-genomics,” Trends in Parasitology, vol. [5] A. Fenwick and J. P. Webster, “Schistosomiasis: challenges for 23, no. 1, pp. 14–20, 2007. control, treatment and drug resistance,” Current Opinion in [24] P. J. Brindley, M. Mitreva, E. Ghedin, and S. Lustigman, Infectious Diseases, vol. 19, no. 6, pp. 577–582, 2006. “Helminth genomics: the implications for human health,” [6] World Health Organization, Weekly Epidemiological Record, PLoS Neglected Tropical Diseases, vol. 3, no. 10, article e538, WHO, 87th edition, 2012. 2009. [7] World Health Organization, 2012, http://www.who.int/health [25] S. P. Lawton, H. Hirai, J. E. Ironside, D. A. Johnston, and D. info/global burden disease/estimates regional 2002 original/ Rollinson, “Genomes and geography: genomic insights into en/. the evolution and phylogeography of the genus Schistosoma,” [8] G. N. Gobert, “Applications for profiling the schistosome tran- Parasites & Vectors, vol. 4, article 131, 2011. scriptome,” Trends in Parasitology, vol. 26, no. 9, pp. 434–439, [26] A. Waeschenbach, B. L. Webster, and D. T. J. Littlewood, 2010. “Adding resolution to ordinal level relationships of tapeworms [9] M. M. Mourao,˜ C. Grunau, P. T. Loverde, M. K. Jones, and G. (Platyhelminthes: Cestoda) with large fragments of mtDNA,” Oliveira, “Recent advances in Schistosoma genomics,” Parasite Molecular Phylogenetics and Evolution, vol. 63, no. 3, pp. 834– Immunology, vol. 34, no. 2-3, pp. 151–162, 2012. 847, 2012. [10] M. T. Swain, D. M. Larkin, C. R. Caffrey et al., “Schistosoma [27] M. Z. Zarowiecki, T. Huyse, and D. T. J. Littlewood, “Making comparative genomics: integrating genome structure, parasite the most of mitochondrial genomes—markers for phylogeny, Journal of Parasitology Research 9

molecular ecology and barcodes in Schistosoma (Platy- [42] L. F. Andrade, L. A. Nahum, L. G. A. Avelar et al., “Eukaryotic helminthes: Digenea),” International Journal for Parasitology, Protein Kinases (ePKs) of the Helminth Parasite Schistosoma vol. 37, no. 12, pp. 1401–1418, 2007. mansoni,” BMC Genomics, vol. 12, article 215, 2011. [28] G.-H. Zhao, J. Li, H.-Q. Song et al., “A specific PCR assay for [43] L. G. Avelar, L. A. Nahum, L. F. Andrade, and G. Oliveira, the identification and differentiation of Schistosoma japonicum “Functional diversity of the Schistosoma mansoni tyrosine geographical isolates in mainland China based on analysis kinases,” Journal of Signal Transduction, vol. 2011, Article ID of mitochondrial genome sequences,” Infection, Genetics and 603290, 11 pages, 2011. Evolution, vol. 12, no. 5, pp. 1027–1036, 2012. [44]L.Anderson,R.J.Pierce,andS.Verjovski-Almeida,“Schis- [29] J. R. Stothard, H. Ameri, I. S. Khamis, L. Blair, U. S. Nyandindi, tosoma mansoni histones: from transcription to chromatin R. A. Kane et al., “Parasitological and malacological surveys regulation; An in silico analysis,” Molecular and Biochemical reveal urogenital Schistosomiasis on Mafia Island, Tanzania to Parasitology, vol. 183, no. 2, pp. 105–114, 2012. be an imported infection,” Acta Tropica. In press. [45] L. L. Silva, M. Marcet-Houben, A. Zerlotini, T. Gabaldon,´ [30] D.T.J.Littlewood,A.E.Lockyer,B.L.Webster,D.A.Johnston, G. Oliveira, and L. A. Nahum, “Evolutionary histories of and T. H. Le, “The complete mitochondrial genomes of expanded peptidase families in Schistosoma mansoni,” Memo- Schistosoma haematobium and Schistosoma spindale and the rias do Instituto Oswaldo Cruz, vol. 106, no. 7, pp. 864–877, evolutionary history of mitochondrial genome changes among 2011. parasitic flatworms,” Molecular Phylogenetics and Evolution, [46]W.Wu,E.G.Niles,H.Hirai,andP.T.LoVerde,“Evolution vol. 39, no. 2, pp. 452–467, 2006. of a novel subfamily of nuclear receptors with members that [31] T. H. Le, D. Blair, and D. P. McManus, “Mitochondrial each contain two DNA binding domains,” BMC Evolutionary genomes of parasitic flatworms,” Trends in Parasitology, vol. Biology, vol. 7, article 27, 2007. 18, no. 5, pp. 206–213, 2002. [47] L. Otero, M. Bonilla, A. V. Protasio, C. Fernandez,´ V. N. [32]T.H.Le,P.F.Humair,D.Blair,T.Agatsuma,D.T.J.Lit- Gladyshev, and G. Salinas, “Thioredoxin and glutathione tlewood, and D. P. McManus, “Mitochondrial gene content, systems differ in parasitic and free-living platyhelminths,” arrangement and composition compared in African and Asian BMC Genomics, vol. 11, no. 1, article 237, 2010. schistosomes,” Molecular and Biochemical Parasitology, vol. [48] C. J. Standley and J. R. Stothard, “DNA barcoding of Schisto- 117, no. 1, pp. 61–71, 2001. soma cercariae reveals a novel sub-lineage within Schistosoma [33] Y. Valles` and J. L. Boore, “Lophotrochozoan mitochondrial rodhaini from Ngamba Island Chimpanzee Sanctuary, Lake genomes,” Integrative and Comparative Biology, vol. 46, no. 4, Victoria,” Journal of Parasitology, vol. 98, no. 5, pp. 1049–1051, pp. 544–557, 2006. 2012. [34] S. W. Attwood, “Studies on the parasitology, phylogeography [49] B. L. Webster, C. L. Culverwell, I. S. Khamis, K. A. and the evolution of host-parasite interactions for the snail Mohammed, D. Rollinson, and J. R. Stothard, “DNA bar- intermediate hosts of medically important trematode genera coding of Schistosoma haematobium on Zanzibar reveals in Southeast Asia,” Advances in Parasitology, vol. 73, pp. 405– substantial genetic diversity and two major phylogenetic 440, 2010. groups,” Acta Tropica. In press. [35] B. L. Webster, V. R. Southgate, D. Timothy, and J. Littlewood, [50] G. R. Franco, A. F. Valadao,˜ V. Azevedo, and E. M. L. Rabelo, “A revision of the interrelationships of Schistosoma including “The Schistosoma gene discovery program: state of the art,” the recently described Schistosoma guineensis,” International International Journal for Parasitology, vol. 30, no. 4, pp. 453– Journal for Parasitology, vol. 36, no. 8, pp. 947–955, 2006. 463, 2000. [36] M. L. Steinauer, I. N. Mwangi, G. M. Maina et al., “Inter- [51] S. Verjovski-Almeida, R. DeMarco, E. A. L. Martins et al., actions between natural populations of human and rodent “Transcriptome analysis of the acoelomate human parasite schistosomes in the lake victoria region of Kenya: a molecular Schistosoma mansoni,” Nature Genetics, vol. 35, no. 2, pp. 148– epidemiological approach,” PLoS Neglected Tropical Diseases, 157, 2003. vol. 2, no. 4, article e222, 2008. [52] E. P. B. Ojopi, P. S. L. Oliveira, D. N. Nunes et al., “A quan- [37] T. Agatsuma, “Origin and evolution of Schistosoma japon- titative view of the transcriptome of Schistosoma mansoni icum,” Parasitology International, vol. 52, no. 4, pp. 335–340, adult-worms using SAGE,” BMC Genomics, vol. 8, article 186, 2003. 2007. [38]J.A.T.Morgan,R.J.Dejong,S.D.Snyder,G.M.Mkoji, [53] D. L. Williams, A. A. Sayed, J. Bernier et al., “Profiling Schis- andE.S.Loker,“Schistosoma mansoni and Biomphalaria: past tosoma mansoni development using serial analysis of gene history and future trends,” Parasitology, vol. 123, supplement, expression (SAGE),” Experimental Parasitology, vol. 117, no. 3, pp. S211–S228, 2001. pp. 246–258, 2007. [39] D. Rollinson, A. Kaukas, D. A. Johnston, A. J. G. Simpson, [54] J. M. Fitzpatrick and K. F. Hoffmann, “Dioecious Schistosoma and M. Tanaka, “Some molecular insights into schistosome mansoni express divergent gene repertoires regulated by evolution,” International Journal for Parasitology, vol. 27, no. pairing,” International Journal for Parasitology, vol. 36, no. 10- 1, pp. 11–28, 1997. 11, pp. 1081–1089, 2006. [40] L. Despres, D. Imbert-Establet, C. Combes, and F. Bonhomme, [55] L. Moertel, D. P. McManus, T. J. Piva, L. Young, R. L. McInnes, “Molecular evidence linking hominid evolution to recent and G. N. Gobert, “Oligonucleotide microarray analysis of radiation of schistosomes (Platyhelminthes: Trematoda),” strain- and gender-associated gene expression in the human Molecular Phylogenetics and Evolution, vol. 1, no. 4, pp. 295– blood fluke, Schistosoma japonicum,” Molecular and Cellular 304, 1992. Probes, vol. 20, no. 5, pp. 280–289, 2006. [41] L. A. Nahum and S. L. Pereira, “Phylogenomics, protein family [56] M. Waisberg, F. P. Lobo, G. C. Cerqueira et al., “Schistosoma evolution, and the tree of life: an integrated approach between mansoni: microarray analysis of gene expression induced by molecular evolution and computational intelligence,” Studies host sex,” Experimental Parasitology, vol. 120, no. 4, pp. 357– in Computational Intelligence, vol. 122, pp. 259–279, 2008. 363, 2008. 10 Journal of Parasitology Research

[57] R. DeMarco, K. C. Oliveira, T. M. Venancio, and S. Verjovski- [73] P. J. Skelly, A. Da’dara, and D. A. Harn, “Suppression of Almeida, “Gender biased differential alternative splicing pat- cathepsin B expression in Schistosoma mansoni by RNA terns of the transcriptional cofactor CA150 gene in Schisto- interference,” International Journal for Parasitology, vol. 33, no. soma mansoni,” Molecular and Biochemical Parasitology, vol. 4, pp. 363–369, 2003. 150, no. 2, pp. 123–131, 2006. [74] M. M. Mourao,˜ N. Dinguirard, G. R. Franco, and T. P.Yoshino, [58] E. R. Jolly, C. S. Chin, S. Miller et al., “Gene expression “Phenotypic screen of early-developing larvae of the blood patterns during adaptation of a helminth parasite to different fluke, Schistosoma mansoni, using RNA interference,” PLoS environmental niches,” Genome Biology, vol. 8, no. 4, article Neglected Tropical Diseases, vol. 3, no. 8, article e502, 2009. R65, 2007. [75] S. Stefanic,ˇ J. Dvorˇak,´ M. Horn et al., “RNA interference in [59] J. M. Fitzpatrick, E. Peak, S. Perally et al., “Anti-schistosomal Schistosoma mansoni schistosomula: selectivity, sensitivity and intervention targets identified by lifecycle transcriptomic operation for larger-scale screening,” PLoS Neglected Tropical analyses,” PLoS Neglected Tropical Diseases, vol. 3, no. 11, Diseases, vol. 4, no. 10, article e850, 2010. article e543, 2009. [76] P. Geldhof, A. Visser, D. Clark et al., “RNA interference in [60] P. Driguez, D. L. Doolan, A. Loukas, P. L. Felgner, and D. parasitic helminths: current situation, potential pitfalls and P. McMa nu s , “ Schistosomiasis vaccine discovery using im- future prospects,” Parasitology, vol. 134, no. 5, pp. 609–619, munomics,” Parasites and Vectors, vol. 3, no. 1, article 4, 2010. 2007. [61] H. E. G. McWilliam, P. Driguez, D. Piedrafita, D. P. McManus, [77]J.M.Correnti,P.J.Brindley,andE.J.Pearce,“Long-term and E. N. T. Meeusen, “Novel immunomic technologies for suppression of cathepsin B levels by RNA interference retards schistosome vaccine development,” Parasite Immunology, vol. schistosome growth,” Molecular and Biochemical Parasitology, 34, no. 5, pp. 276–284, 2012. vol. 143, no. 2, pp. 209–215, 2005. [62]G.N.Gobert,D.P.McManus,S.Nawaratna,L.Moertel, [78] M. E. Morales, G. Rinaldi, G. N. Gobert, K. J. Kines, J. F. Tort, J. Mulvenna, and M. K. Jones, “Tissue specific profiling and P. J. Brindley, “RNA interference of Schistosoma mansoni of females of Schistosoma japonicum by integrated laser cathepsin D, the apical enzyme of the hemoglobin proteolysis microdissection microscopy and microarray analysis,” PLoS cascade,” Molecular and Biochemical Parasitology, vol. 157, no. Neglected Tropical Diseases, vol. 3, no. 6, article e469, 2009. 2, pp. 160–168, 2008. [63] S. S. K. Nawaratna, D. P. McManus, L. Moertel, G. N. [79] G. Rinaldi, M. E. Morales, Y. N. Alrefaei et al., “RNA Gobert, and M. K. Jones, “Gene atlasing of digestive and interference targeting leucine aminopeptidase blocks hatching reproductive tissues in Schistosoma mansoni,” PLoS Neglected of Schistosoma mansoni eggs,” Molecular and Biochemical Tropical Diseases, vol. 5, no. 4, Article ID e1043, 2011. Parasitology, vol. 167, no. 2, pp. 118–126, 2009. [80] B. E. Swierczewski and S. J. Davies, “A schistosome cAMP- [64] M. Hope, M. Duke, and D. P. McManus, “A biological dependent protein kinase catalytic subunit is essential for and immunological comparison of Chinese and Philippine parasite viability,” PLoS Neglected Tropical Diseases, vol. 3, no. Schistosoma japonicum,” International Journal for Parasitology, 8, article e505, 2009. vol. 26, no. 3, pp. 325–332, 1996. [81]R.S.Kasinathan,W.M.Morgan,andR.M.Greenberg,“Ge- [65]G.T.Almeida,M.S.Amaral,F.C.F.Beckedorff,J.P. netic knockdown and pharmacological inhibition of parasite Kitajima, R. DeMarco, and S. Verjovski-Almeida, “Exploring multidrug resistance transporters disrupts EGG production in the Schistosoma mansoni adult male transcriptome using Schistosoma mansoni,” PLoS Neglected Tropical Diseases, vol. 5, RNA-seq,” Experimental Parasitology, vol. 132, no. 1, pp. 22– no. 12, Article ID e1425, 2011. 31, 2011. [82] Y. Yang, Y. Jin, P. Liu et al., “RNAi silencing of type v collagen [66] S. Verjovski-Almeida and R. DeMarco, “Gene structure and in Schistosoma japonicum affects parasite morphology, spawn- splicing in schistosomes,” Journal of Proteomics,vol.74,no.9, ing, and hatching,” Parasitology Research, vol. 111, no. 3, pp. pp. 1515–1518, 2011. 1251–1257, 2012. [67] K. E. M. Hastings, “SL trans-splicing: easy come or easy go?” [83] G. Rinaldi, T. I. Okatcha, A. Popratiloff et al., “Genetic Trends in Genetics, vol. 21, no. 4, pp. 240–247, 2005. manipulation of Schistosoma haematobium, the neglected [68] G. Cheng, L. Cohen, D. Ndegwa, and R. E. Davis, “The flat- Schistosome,” PLoS Neglected Tropical Diseases, vol. 5, no. 10, worm spliced leader 3-terminal AUG as a translation initiator Article ID e1348, 2011. methionine,” The Journal of Biological Chemistry, vol. 281, no. [84]M.H.Tran,T.C.Freitas,L.Cooperetal.,“Suppression 2, pp. 733–743, 2006. of mRNAs encoding tegument tetraspanins from Schisto- [69] R. E. Davis, C. Hardwick, P. Tavernier, S. Hodgson, and H. soma mansoni results in impaired tegument turnover,” PLoS Singh, “RNA trans-splicing in flatworms. Analysis of trans- Pathogens, vol. 6, no. 4, Article ID e1000840, 2010. spliced mRNAs and genes in the human parasite, Schistosoma [85] A. A. Sayed, S. K. Cook, and D. L. Williams, “Redox balance mansoni,” The Journal of Biological Chemistry, vol. 270, no. 37, mechanisms in Schistosoma mansoni rely on peroxiredoxins pp. 21813–21819, 1995. and albumin and implicate peroxiredoxins as novel drug [70] R. E. Davis and S. Hodgson, “Gene linkage and steady targets,” The Journal of Biological Chemistry, vol. 281, no. 25, state RNAs suggest trans-splicing may be associated with a pp. 17001–17010, 2006. polycistronic transcript in Schistosoma mansoni,” Molecular [86] A. N. Kuntz, E. Davioud-Charvet, A. A. Sayed et al., “Thiore- and Biochemical Parasitology, vol. 89, no. 1, pp. 25–39, 1997. doxin glutathione reductase from Schistosoma mansoni:an [71] P. J. Brindley and E. J. Pearce, “Genetic manipulation of essential parasite enzyme and a key drug target,” PLoS schistosomes,” International Journal for Parasitology, vol. 37, Medicine, vol. 4, no. 6, Article ID e206, 2007. no. 5, pp. 465–473, 2007. [87] M. D. M. Mourao,˜ N. Dinguirard, G. R. Franco, and T. P. [72] J. P. Boyle, X. J. Wu, C. B. Shoemaker, and T. P. Yoshino, Yoshino, “Role of the endogenous antioxidant system in the “Using RNA interference to manipulate endogenous gene protection of Schistosoma mansoni primary sporocysts against expression in Schistosoma mansoni sporocysts,” Molecular and exogenous oxidative stress,” PLoS Neglected Tropical Diseases, Biochemical Parasitology, vol. 128, no. 2, pp. 205–215, 2003. vol. 3, no. 11, article e550, 2009. Journal of Parasitology Research 11

[88] M. A. Ayuk, S. Suttiprapa, G. Rinaldi, V. H. Mann, C. M. Lee, and P. J. Brindley, “Schistosoma mansoni U6 gene promoter- driven short hairpin RNA induces RNA interference in human ﬁbrosarcoma cells and schistosomules,” International Journal for Parasitology, vol. 41, no. 7, pp. 783–789, 2011. [89] R. Duvoisin, M. A. Ayuk, G. Rinaldi et al., “Human U6 promoter drives stronger shRNA activity than its schistosome orthologue in Schistosoma mansoni and human ﬁbrosarcoma cells,” Transgenic Research, vol. 21, no. 3, pp. 511–521, 2012. [90] G. Rinaldi, S. Suttiprapa, J. F. Tort, A. E. Folley, D. E. Skinner, and P. J. Brindley, “An antibiotic selection marker for schistosome transgenesis,” International Journal for Parasitology, vol. 42, no. 1, pp. 123–130, 2012. [91] S. Beckmann and C. G. Grevelding, “Paving the way for transgenic schistosomes,” Parasitology, vol. 139, no. 5, pp. 651–668, 2012. [92] G. Rinaldi, S. E. Eckert, I. J. Tsai et al., “Germline transgenesis and insertional mutagenesis in Schistosoma mansoni mediated by murine leukemia virus,” PLoS Pathogens, vol. 8, no. 7, Article ID e1002820, 16 pages, 2012. [93] A. Zerlotini, M. Heiges, H. Wang et al., “SchistoDB: a Schisto- soma mansoni genome resource,” Nucleic Acids Research, vol. 37, no. 1, pp. D579–D582, 2009. [94] S. Abubucker, J. Martin, C. M. Taylor, and M. Mitreva, “Helm- CoP: an online resource for Helminth functional genomics and drug and vaccine targets prioritization,” PLoS ONE, vol. 6, no. 7, Article ID e21832, 2011. [95] F. J. Logan-Klumpler, N. De Silva, U. Boehme et al., “GeneDB- an annotation database for pathogens,” Nucleic Acids Research, vol. 40, no. D1, pp. D98–D108, 2012. Hindawi Publishing Corporation Journal of Parasitology Research Volume 2012, Article ID 520308, 10 pages doi:10.1155/2012/520308

Clinical Study Changes in T-Cell and Monocyte Phenotypes In Vitro by Schistosoma mansoni Antigens in Cutaneous Leishmaniasis Patients

Aline Michelle Barbosa Baﬁca,1 Luciana Santos Cardoso,1, 2 Sergio´ Costa Oliveira,3, 4 Alex Loukas,5 Alfredo Goes,´ 3 RicardoRiccioOliveira,1 Edgar M. Carvalho,1, 4, 6 and Maria Ilma Araujo1, 4, 6

1 Serviço de Imunologia, Complexo Hospitalar Universitario´ Professor Edgard Santos, Universidade Federal da Bahia, 5RuaJoao˜ das Botas s/n, Canela, 40110-160 Salvador, BA, Brazil 2 Departamento de Ciências da Vida, Universidade do Estado da Bahia, 2555 Rua Silveira Martins, Cabula, 41.150-000 Salvador, BA, Brazil 3 Departamento de Bioqu´ımica e Imunologia, Instituto de Ciências Biologicas,´ Universidade Federal de Minas Gerais, 6627 Avenida Antonioˆ Carlos, Pampulha, 31270-901 Belo Horizonte, MG, Brazil 4 Instituto Nacional de Ciência e Tecnologia em Doenças Tropicais (INCT-DT-CNPQ/MCT), Rua Joao˜ das Botas s/n, Canela, 40110-160 Salvador, BA, Brazil 5 Queensland Tropical Health Alliance and School of Public Health and Tropical Medicine, James Cook University, Cairns, QLD 4878, Australia 6 Escola Bahiana de Medicina e Saude´ Publica,´ No. 275 Avenida Dom Joao˜ VI, Brotas, 40290-000 Salvador, BA, Brazil

Correspondence should be addressed to Aline Michelle Barbosa Baﬁca, alinebaﬁ[email protected]

Received 13 July 2012; Revised 24 September 2012; Accepted 8 October 2012

Academic Editor: Andrea Teixeira-Carvalho

Copyright © 2012 Aline Michelle Barbosa Baﬁca et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

High levels of proinflammatory cytokines such as IFN-γ and TNF are associated with tissue lesions in cutaneous leishmaniasis (CL). We previously demonstrated that Schistosoma mansoni antigens downmodulate the in vitro cytokine response in CL. In the current study we evaluated whether S. mansoni antigens alter monocyte and T-lymphocyte phenotypes in leishmaniasis. Peripheral blood mononuclear cells of CL patients were cultured with L. braziliensis antigen in the presence or absence of the S. mansoni antigens rSm29, rSmTSP-2- and PIII. Cells were stained with fluorochrome conjugated antibodies and analyzed by flow cytometry. The addition of rSm29 to the cultures decreased the expression of HLA-DR in nonclassical (CD14+CD16++) monocytes, while the addition of PIII diminished the expression of this molecule in classical (CD14++CD16−) and intermediate (CD14++CD16+) monocytes. The addition of PIII and rSmTSP-2 resulted in downmodulation of CD80 expression in nonclassical and CD86 expression in intermediate monocytes, respectively. These two antigens increased the expression of CTLA-4 in CD4+ T cells and they also expanded the frequency of CD4+CD25highFoxp3+ T cells. Taken together, we show that S. mansoni antigens, mainly rSmTSP-2 and PIII, are able to decrease the activation status of monocytes and also to upregulate the expression of modulatory molecules in T lymphocytes.

1. Introduction Tegumentary leishmaniasis presents with a wide spectrum of clinical manifestations ranging from localized skin to American tegumentary leishmaniasis is a disease caused by widespread mucocutaneous lesions depending on the par- parasites of the genus Leishmania. This disease represents asite species [2] and the host immune response [3, 4]. T a significative public health problem worldwide with an cell-mediated immunity is crucial to host protection against incidence of 1.5 million new cases in recent years [1]. Leishmania sp. infection; however skin and mucosal lesions 2 Journal of Parasitology Research occurs due to a deregulated T helper 1 (Th1) cell response CL patients, the frequency of CD14+CD16+ monocytes was with high production of proinflammatory cytokines such as significantly higher compared to healthy controls and they IFN-γ and TNF [3, 4]. were positively correlated with the lesion size [22]. This Experimental studies have shown that Schistosoma man- subtype of monocytes is considered as antigen presenting soni infection or parasite products, by inducing regulatory cell [16], which produces cytokines and might be the most cells and cytokines, are able to prevent some Th1-mediated important subtype in activating T cells response. autoimmune diseases in mice such as type I diabetes, The activation of T cells requires the antigen recognition experimental autoimmune encephalomyelitis, and psoriasis in the MHC context and also signaling given by co- [5–7]. Recently, we demonstrated that the recombinant S. stimulatory molecules which interact with corresponding mansoni antigens Sm29 SmTSP-2, and also PIII down- ligands on antigen presenting cells (APC). One of the most modulated the production of IFN-γ and TNF in a group important co-stimulatory molecules on T cells is CD28, of cutaneous leishmaniasis (CL) patients [8]. In the present which is constitutively expressed and binds to CD80 and study, these antigens were tested regarding their ability to CD86 on the APC. CD86 is constitutively expressed at low alter the monocyte and lymphocyte profiles. Studies have levels and it is rapidly upregulated after primary antigen shown that Sm29 and SmTSP-2 antigens are secreted by the recognition, whereas CD80 exhibits delayed expression membrane and/or tegument of the S. mansoni adult worm. kinetics [23]. Other ligand for CD80 and CD86 is CTLA-4. Proteins secreted or localized on the surface of Schistosoma The expression of this molecule is rapidly upregulated after spp., which are in intimate contact with host tissues, might T cell activation and provides a negative signal limiting the be more effective in triggering immunoregulatory processes immune response [24–26]. In vitro study has shown that the [9]. The Sm29 is a membrane-bound glycoprotein located on addition of CTLA-4-Ig to block CD28-B7 interaction in CL the tegument of the adult worm and lung stage schistosomula patients PBMC cultures stimulated with Leishmania antigen [10]. SmTSP-2 is a recombinant protein (tetraspanin) from led to a downmodulation of IFN-γ and TNF secretion [27]. S. mansoni tegument. In a mice model, immunization with Other similar study showed that blocking the co-stimulatory SmTSP-2 resulted in a 57% reduction in adult worm burdens molecules CD80 and CD86 in human macrophages from and a 64% reduction in liver egg burdens compared with Leishmania-naive donors infected with L. major resulted in control animals [11]. PIII is a multivalent antigen obtained a significant reduction in IFN-γ, IL-5, and IL-12 production from S. mansoni adult worms that modulates granuloma size [28]. in mice infected with the parasite [12, 13]. These antigens In this study we evaluated whether S. mansoni antigens have been evaluated by our group regarding their potential to alter the expression of CD80 and CD86 on monocytes of CL induce IL-10 production and suppress Th2 response in vitro patients and also the expression of CTLA-4 in T lymphocytes. in cells of asthmatic individuals [14]. Additionally, we accessed the frequency of regulatory T cells Together with the Th1 immune response, monocytes induced by S. mansoni antigens when they were added to the and macrophage are key cells in controlling Leishmania sp. cell cultures stimulated with Leishmania antigens. infection. Monocytes have been classifying into different The CD4+CD25+ regulatoryTcellshavebeenextensively subpopulations in mice models and also in humans. A studied due to their critical function in maintaining self- new nomenclature was recently published defining human tolerance. These cells can express both low and high CD25 monocytes into three subtypes. The major population of levels in mice models; however, only the CD4+CD25high human monocytes (90%) presents high expression of CD14 population exhibits a strong regulatory function in humans. and lack of expression of CD16 (CD14++CD16−). They are These CD4+CD25high T cells also express FOXP3, a molecule referred to as classical monocytes. Intermediate monocytes associated with regulatory functions [29]. This T cell subset are those which express CD14 and low CD16 expression in humans comprises ∼1.5–3% of circulating CD4+ T cells. (CD14++CD16+), while the nonclassical monocytes express They inhibit proliferation and cytokine secretion induced CD16 and relatively low CD14 (CD14+CD16++)[15]. In by TCR cross-linking of CD4+CD25− responder T cells in a study conducted by Wong et al. [16] the characteris- a contact-dependent manner [30] and completely abrogate tics of classical, intermediate and, nonclassical monocytes IL-2-dependent proliferation of NK cells [31]. Moreover, were evaluated through the gene expression profiling. They regulatory T cells are able to downregulate the intensity observed that classical monocytes express genes involved and duration of both Th1 and Th2 immune responses in in angiogenesis, wound healing, and coagulation, being infectious diseases limiting damage to self-tissue [32, 33]. involved in tissue repair functions in addition to high Our hypothesis in the present study is that the pathology expression of proinflammatory genes [16]; intermediate of cutaneous leishmaniasis results from monocyte and T monocytes highly express MHC class II indicating they cell hypersensitivity due to impaired regulatory mechanisms. have antigen presenting cell function and T cell stimulatory In this context, the use of S. mansoni antigens which properties. Nonclassical monocytes express genes involved in induce regulatory cells and molecules would prevent the cytoskeleton rearrangement, which may be responsible for its inflammatory process. To test this hypothesis we evaluated high motility observed in vivo [17]. whether the addition of S. mansoni antigens to cell cultures A high frequency of CD16+ monocyte subsets have of leishmaniasis patients would modify the phenotype and been demonstrated in Mycobacterium tuberculosis infection activation status of monocytes and lymphocytes. Specifically, and in viral infections such as hepatitis B (HBV) [18], we evaluated the expression of HLA-DR, CD80, and CD86 hepatitis C (HCV) [19], HIV [20], and Dengue [21]. In on classical, intermediate, and nonclassical monocytes and Journal of Parasitology Research 3

CD28, CTLA-4, CD25, and Foxp3 in T lymphocytes from [11, 36]. The proteins rSm29 and PIII were provided by the CL patients in response to the soluble Leishmania braziliensis Institute of Biological Science, Department of Biochemistry antigen (SLA) in the presence or absence of the S. mansoni and Immunology, UFMG, Brazil. antigens rSm29, rSmTSP-2, and PIII. 2.3. Cell Culture and Intracellular and Surface Staining. 2. Materials and Methods Intracellular and surface molecules were evaluated by flow cytometry (FACSort, BD Biosciences, San Jose, CA). In 2.1. Patients and the Endemic Area. The study included order to perform the intracellular staining, peripheral blood the first 30 cutaneous leishmaniasis patients living in the mononuclear cells (PBMC, 3 × 105) obtained through the endemic area of tegumentary leishmaniasis, Corte de Pedra, Ficoll-Hypaque gradient were cultured in vitro with SLA Bahia, Brazil, who attended the local Health Post from March (5 μg/mL) in the presence or absence of the recombinant 2010 to March 2012 and agreed in participate. Corte de S. mansoni antigens Sm29, SmTSP-2, and PIII (5 μg/mL) ◦ Pedra is located in the southeast region of the State of Bahia, for 20 h, 37 C, and 5% of CO2. During the last 4 h of Brazil which is well known for its high rate of L. braziliensis culture, Brefeldin A (10 μg/mL; Sigma, St. Louis, MO), transmission. which impairs protein secretion by the Golgi complex, was The diagnostic of cutaneous leishmaniasis was made added to the cultures. Afterwards, the cells were washed considering a clinical picture characteristic of CL, parasite in PBS and fixed in 4% formaldehyde for 20 min at room isolation or a positive delayed-type hypersensitivity (DTH) temperature. Specific staining was performed with cychrome response to Leishmania antigen, and a histological feature of (CY)-labeled antibody conjugated with anti-CTLA-4 mAbs CL. in saponin buffer (PBS, supplemented with 0.5% BSA and The inclusion criteria to this study were patients with 0.5% saponin) and phycoerythrin (PE)-labeled antibody 5 to 60 years of age diagnosis of CL with the presence of conjugated with anti-Foxp3 in Foxp3 staining buffer set active skin lesions. The exclusion criteria were pregnancy, (eBioscience). For double or triple staining, mAbs to human chronic diseases such as diabetes and asthma, and also HIV CD4, CD8, and/or CD25 conjugated with FITC or CY were and HTLV-1 infection. used. Immunological analyses were performed prior to the The evaluation of co-stimulatory molecule expression specific therapy to leishmaniasis for all patients. There were was performed in PBMCs stimulated for 60 h with the not enough cells to perform the whole experiment every time same antigens aforementioned. Cells were incubated with since they require a larger number of cells more than what fluorescein isothiocyanate (FITC)-, PE-, or CY-labeled anti- could be obtained from some patients. body solutions for 20 min at 4◦Cinavolumeof20μLin The frequency of helminth infection in the leishmaniasis PBS. After staining, preparations were washed with 0.1% endemic area of Corte de Pedra is 88.3% and S. mansoni sodium azide PBS, fixed with 200 μL of 4% formaldehyde infection presents in 16.7% of cutaneous leishmaniasis in PBS and kept at 4◦C. The antibodies used for the patients. staining were immunoglobulin isotype controls-FITC, PE The Ethical Committee of the Maternidade Climerio´ de and CY, anti-CD4-FITC, anti-CD8-FITC, anti-CD25-FITC, Oliveira, Federal University of Bahia approved the present anti-CD14-FITC, CD16-CY, anti-CD4-CY and anti-CD8- study, and an informed consent was obtained from all CY, anti-CD80-PE, anti-CD86-PE, anti-HLA-DR-PE, anti- participants or their legal guardians. CD28-PE (all from BD Biosciences Pharmingen). A total of 50,000 and 100,000 events were acquired for surface and 2.2. Antigen Preparation. The soluble Leishmania antigen intracellular experiments, respectively. used in this study was obtained from a Leishmania lysate (crude antigen). It was prepared from a L. braziliensis strain 2.4. Analysis of FACS Data. The frequency of positive cells (MHOM/BR/2001) as previously described [34]. was analyzed by flow cytometry using the program flowjo The S. mansoni antigens used in this study included in two regions. The lymphocyte region was determined Sm29, a Schistosoma mansoni recombinant protein [10]; using granularity (SSC) × size (FSC) plot. Monocytes were SmTSP-2, a recombinant protein (tetraspanin) from S. selected based on their granularity and expression of CD14 mansoni tegument [11];afractionofS. mansoni soluble adult and CD16. Limits for the quadrant markers were always worm antigen (SWAP) obtained by anionic chromatography set based on negative populations and isotype controls. A (FPLC), called PIII. The Sm29 was cloned in E. coli and representative density graph of one experiment showing tested for lipopolysaccharide (LPS) contamination using a lymphocyte and monocyte regions is shown in Figures 1(a) commercially available LAL Chromogenic Kit (CAMBREX). and 1(b),respectively. ThelevelsofLPSinSm29werebelow0.25ng/mL.However inordertoneutralizepotentialeffects of LPS presented in low levels in the S. mansoni recombinant antigen, Polymyxin 2.5. Statistical Analyses. Statistical analyses were performed B was added to cell cultures every 12 hours according using the software GraphPad Prism (GraphPad Software, San to the established protocol [35]. The SmTSP-2 used in Diego, CA). The frequency of positive cells was expressed this study was produced in Pichia pastoris fermentation as percentages and the intensity of expression as mean cultures and it has been kindly provided by Dr. Alex Loukas intensity fluorescence (MFI). The differences between means 4 Journal of Parasitology Research

4 1K 10 Nonclassical

800 103 Intermediate 600 102

400 SSC-H:: SSC-heightSSC-H:: FL3-H:: CD16PE-Cy5 FL3-H:: 101 200

Classical 0 100 0 200 400 600 800 1K 100 101 102 103 104 FSC-H:: FSC-height FL1-H:: CD14 FITC (a) (b)

Figure 1: Strategy for T cell and monocytes evaluation by flow cytometry. The cell populations were defined by nonspecific fluorescence from the forward (FSC) and side scatter (SSC) as parameters of cell size and granularity, respectively. Lymphocytes region was determined using SSC × FSC plot (G1) and monocytes region (G2) were selected based on their granularity and expression of CD14 (a). (b) represents the strategy for monocytes subsets classification through the expression of CD14 and CD16. Representative graph of one experiment.

Table 1: Demographic characteristics of the cutaneous leishmania- rSm29 and PIII to the cultures expanded the frequency of sis patients included in the study. nonclassical (CD14+CD16++)(mean± SEM = 7.4 ± 1.2% and 8.1 ± 1.9%, resp.) compared to cultures stimulated with Characteristics (n = 30) Values SLA alone (5.7 ± 0.9%; P<0.05. Figure 2(A)). We also ± . ± . Age, mean SD, years 29 1 11 8 observed that the frequency of classical monocytes was Sex, female/male 13/17 higher in cultures without S. mansoni antigens (Figure 2(A)). ◦ N of lesion Moreover, in the presence of PIII there was a reduction in 1, no. (%) 24 (80.0) the expression of HLA-DR in classical (CD14++CD16−)and ≥2, no. (%) 06 (20.0) intermediate (CD14++CD16+) monocytes (496±72 MFI and Size of lesion, median mm2 (IQR) 130 (66.5–342) 544 ± 78.6MFI,resp.)comparedtoculturesstimulatedwith IQR: interquartile range; SD: standard deviation. SLA alone (611 ± 91 MFI and 771 ± 128 MFI, respectively; P<0.05. Figure 2(B)). There was also a reduction in HLA-DR expression on CD14+CD16++ monocytes in the were assessed using Wilcoxon matched pairs test. Statistical presence of rSm29 (547 ± 140.5 MFI) in comparison to significance was established at the 95% confidence interval. SLA alone (718 ± 188.4MFI;P<0.05. Figure 2(B)). Additionally, the expression of HLA-DR was higher in 3. Results classical and intermediate monocytes in the presence of SLA or SLA plus S. mansoni antigens compared to nonstimulated A total of 30 patients with cutaneous leishmaniasis were cultures. enrolled in this study, 17 were male and 13 were female, The expression of CD80 was also reduced in nonclassical with a mean age of 29.1 ± 11.8 years (range 6–48 years). monocytes compared to cultures stimulated with SLA. The The majority of patients presented with a single lesion (80%) addition of PIII to the cultures also resulted in reduced and the median lesions size was 130 mm2 (IQR, 66.5–342; expression of the co-stimulatory molecule CD80 on Table 1). nonclassical monocytes (61.2 ± 19.5MFI)comparedtocul- tures without S. mansoni antigens (82.3±23.3MFI;P<0.05. 3.1. The Effect of the Addition of S. mansoni Antigens on Mono- Figure 2(C)). Also a decrease in the expression of CD86 on cyte Phenotype and Expression of Co-Stimulatory Molecules. intermediate monocytes from 562 ± 149.7 MFI to 447.8 ± We evaluated the frequency of different monocyte subsets 112.5 MFI was observed when rSmTSP-2 was added to the (classical, intermediate, and nonclassical) and also the cultures (P<0.05; Figure 2(D)). There was no significant expression of co-stimulatory molecules in these monocytes difference in the levels of CD86 expression on monocytes in after in vitro stimulation with SLA in the presence or culture stimulated with SLA after the addition of rSm29 and absence of S. mansoni antigens. The addition of the antigens PIII (Figure 2(D)). Journal of Parasitology Research 5

80 1000 a a 60 750 a b a a b a a a a a b 40 500

20 expression HLA-DR 250 in monocytes (MIF) b b Frequency ofFrequency monocyte (%) 0 0 Classical Intermediate Nonclassical Classical Intermediate Nonclassical (A) (B) 1000 160

750 120 a b 80 500 b CD86 expression CD86 in monocytes (MIF) CD80 expression

in monocytes (MIF) 40 250

0 0 Classical Intermediate Nonclassical Classical Intermediate Nonclassical

WS SLA+rSmTSP-2 WS SLA+rSmTSP-2 SLA SLA+PIII SLA SLA+PIII SLA+rSm29 SLA+rSm29 (C) (D) Figure 2: Subsets of monocytes and co-stimulatory molecules expression in monocytes of CL patients (n = 18) stimulated in vitro with SLA in the presence or absence of S. mansoni antigens rSm29 and rSmTSP-2 or with a fraction of S. mansoni soluble adult worm antigen (SWAP) PIII. Frequency of classical (CD14++CD16−), intermediate (CD14++CD16+) and nonclassical (CD14+CD16++), monocytes (A), and expression of HLA-DR, CD80, and CD86 in classical, intermediate and nonclassical monocytes of cutaneous leishmaniasis patients (B– D). Cells were stained for surface expression of CD14, CD16, HLA-DR, CD80, and CD86 using ﬂow cytometry. aP<0.05 cultures without stimulation (WS) versus SLA + S. mansoni antigens; bP<0.05 SLA versus SLA + S. mansoni antigens (Wilcoxon matched pairs test).

3.2. The Effect of the Addition of S. mansoni Antigens on T the presence of SLA and SLA plus S. mansoni antigens Cell Phenotype and Expression of Co-Stimulatory Molecules. compared to nonstimulated cultures (Figure 3(A)). PBMC of CL patients were incubated with SLA in the pres- We also evaluated the expression of CD28+ on lympho- ence or absence of S. mansoni antigens and the phenotype of cytes in cultures stimulated with SLA with or without the T cells were evaluated. The addition of rSm29 antigen to the addition of S. mansoni antigens. The expression of CD28+ on cultures increased the frequency of CD4+ T cells (mean ± CD8+ T cells was higher in cultures stimulated with rSm29 SEM = 40.8 ± 2.8%) in comparison to cultures with SLA (91 ± 14 MFI, resp.) compared with SLA alone (84 ± 14 alone (34.8 ± 2.8%, P<0.05; Figure 3(A)). There was no MFI; P<0.05. Figure 3(B)). The addition of rSmTSP-2 and significant difference in the frequency of CD4+ T cells after PIII antigens to the cultures did not alter significantly the the addition of rSmTSP-2 or PIII antigens to the cultures expression of CD28 on CD4+ and CD8+ T cells (Figure 3(B)). (37.8 ± 2.7% and 35.8 ± 2.6%, resp.) compared to SLA Regarding the expression of CTLA-4, rSmTSP-2 and PIII alone (34.8 ± 2.8%; Figure 3(A)). Likewise, there was no antigens were able to increase the expression of this molecule significant variation in the frequency of CD8+ T cells by the in CD4+ T cells (58 ± 5MFIand53± 4.6MFI,resp.)incom- presence of rSm29, rSmTSP-2, and PIII antigens (5.9 ± 1.2%, parison with SLA alone (49.6 ± 4MFI,respectively;P<0.05. 4.6±0.8%, and 6.2±1.1%, resp.) to the cultures in relation to Figure 3(C)). The expression of CTLA-4 was also higher the cultures stimulated with SLA without S. mansoni antigens in CD4+ T cells and CD8+ T cells in cultures stimulated (5.7 ± 1.2%; Figure 3(A)). The frequency of CD4+ T cells was with SLA plus rSmTSP-2 or PIII compared to nonstimulated diminished by the presence of SLA and SLA plus rSmTSP-2 cultures (Figure 3(C)). Moreover, the addition of rSmTSP-2 or PIII, while the frequency of CD8+ T cells was higher in and PIII antigens to the cultures expanded the frequency of 6 Journal of Parasitology Research

60 160

b 120 a b 40 a a

20 40 Frequency ofFrequency lymphocyte (%) a a a a in lymphocyteCD28 expression (MFI)

0 0 TCD4+ TCD8+ TCD4+ TCD8+ (A) (B) 100 15

b a b 75 12 a b a

a b a (%) cells + 9 50 Foxp3

high 6

25 CD25 + 3 CD4 CTLA-4 expression in lymphocyte expression (MIF) CTLA-4 0 + + TCD4 TCD8 0

WS SLA+rSmTSP-2 WS SLA+rSmTSP-2 SLA SLA+PIII SLA SLA+PIII SLA+rSm29 SLA+rSm29 (C) (D) Figure 3: Phenotype of T cells and co-stimulatory molecules expression on T lymphocytes of CL patients (n = 17) stimulated in vitro with SLA in the presence or absence of S. mansoni antigens rSm29, rSmTSP-2 or with a fraction of S. mansoni soluble adult worm antigen (SWAP) PIII. Frequency of CD4+ and CD8+ T cells (A), expression of CD28 and CTLA-4 in CD4+ and CD8+ T cells (B and C, resp.) and the frequency of CD4+CD25highFoxp3+ Tcells(n = 13) (D). Cells were stained for surface expression of CD4, CD8, CD25 and CD28, while the expression of CTLA-4 and Foxp3 were evaluated intracellularly using ﬂow cytometry. aP<0.05 cultures without stimulation (WS) versus SLA + S. mansoni antigens; bP<0.05 SLA versus SLA + S. mansoni antigens (Wilcoxon matched pairs test).

CD4+CD25highFoxp3+ T cells (10 ± 2.4% and 10.3 ± 1.9%, release high levels of proinflammatory cytokines and nitro- resp.) compared to cultures with SLA in the absence of gen and oxygen reactive intermediates, leading to a tissue S. mansoni antigens (8.1 ± 1.8%; P<0.05. Figure 3(D)). lesion. The frequency of CD4+CD25highFoxp3+ T cells was also A balance between the proinflammatory response char- higher in cultures stimulated with SLA plus PIII compared acterized by the production of IFN-γ and TNF, and the to nonstimulated cultures (Figure 3(D)). regulatory response with the production of IL-10 has been observed in individuals exposed to the Leishmania braziliensis in endemic areas of leishmaniasis in Brazil. 4. Discussion Those individuals do not develop the disease and they are designated as “subclinical” subjects [37]. The balanced The Th1 immune response associated with macrophage acti- immune response described in these individuals may be the vation and killing of Leishmania sp. is paradoxically related to key to achieving a harmless host-parasite relationship. the development of cutaneous and mucosal leishmaniasis. In There are other evidences that IL-10 is capable of an attempt to control parasite growth, activated macrophages downmodulating the inflammatory response associated with Journal of Parasitology Research 7 human tegumentary leishmaniasis [4, 38–40]. However, The addition of the antigens rSm29 and PIII to the mononuclear cells of mucosal leishmaniasis (ML) patients PBMC cultures stimulated with SLA increased the frequency have a decreased ability to produce this cytokine and to of nonclassical monocytes. This was not expected as a cell respond to IL-10 after in vitro restimulation with L. brazilien- with high mobility and migratory capacity [16, 47], they sis antigen [3]. may contribute to Leishmania metastasis and consequent Recently it has been shown that during chronic Schis- development of more severe forms of the disease, such as the tosoma mansoni infection, cells from the innate immune mucosal and disseminated forms. response, such as monocytes and regulatory T cells produce We found, however, that the S. mansoni antigens used high levels of IL-10 [24, 41–43]. Our group has performed in this study, particularly PIII, diminished the expression studies in an attempt to identify S. mansoni antigens with of HLA-DR as well as the expression of the co-stimulatory regulatory properties that enable them to downregulate molecules B7.1 and B7.2 on different monocyte subsets. the inflammatory process associated with certain immune- A downmodulation of monocyte activation is desirable in mediated diseases. For instance, we have shown that the S. leishmaniasis and may result in a reduced inflammatory mansoni antigens rSm29, rSmTSP-2, and PIII induce IL-10 process. and IL-5 production by PBMC of cutaneous leishmaniasis In this study, we also evaluated the expression of co- patients and that they are able to control the in vitro stimulatory molecules in T cells. We observed a significant inflammatory response in a group of patients, independent increase in the frequency of CD4+ T cells by the presence of of the clinical features, such as number and size of lesions the rSm29 antigen in the cultures. The addition of rSmTSP-2 [8]. In this current study we extended the assessment and PIII antigens to the cultures stimulated with SLA of the potential of the antigens rSm29, rSmTSP-2, and increased the expression of CTLA-4 in CD4+ T cells and also PIII in modifying the immune response during cutaneous expanded the frequency of CD4+CD25highFoxp3+ T cells. We leishmaniasis. Specifically, we evaluated the impact of the previously showed that S. mansoni infection expands CD4+ addition of these antigens to the cell culture stimulated with T cell population of the regulatory profile in S. mansoni- Leishmania antigens on monocyte and lymphocyte profile infected asthmatic individuals [24]. In such allergic disease, and activation status. S. mansoni antigens downmodulate the Th2 exacerbated CD4+ T lymphocytes and monocytes are key cells in the inflammatory response in vitro by inducing the production protection against leishmaniasis; however they have been of IL-10 and the expression of the regulatory molecules, associated to the inflammatory process and tissue lesion in CTLA-4 in T lymphocytes [9, 24, 43]. In a murine model of the cutaneous form of disease [20, 44–46]. In leishmaniasis ovalbumin-induced asthma, inhibition of lung inflammatory and also in some others diseases the role of different subsets process, by S. mansoni eggs or by the S. mansoni antigens, of monocytes in the pathology has been described [47]. Sm22.6, PIII, and rSm29 were associated with an increase in For more than two decades, monocytes have been classified the number of CD4+CD25+Foxp3+ T cells and high levels of into classical (CD14+CD16−) and inflammatory, those which IL-10 production [49, 50]. express the molecule CD16 and produce high levels of TNF- T cell-mediated immunity is fundamental to host pro- α [48]. The frequency of CD14+CD16+ in CL patients was tection against Leishmania sp. and the activation of T cells found to be significantly higher compared to healthy controls depends upon signals from the interaction between the co- and they were positively correlated with the lesion size [22]. stimulatory molecules CD28 to its ligands B7.1 and B7.2 Recently, a new nomenclature has been used to clas- on antigen presenting cells (APCs) [23]. We evaluated the sify human blood monocytes into three subsets: classi- expression of CD28 on lymphocytes in cultures stimulated cal (CD14++CD16−), intermediate (CD14++CD16+), and with SLA with or without the addition of S. mansoni nonclassical (CD14+CD16++)[15]. The classical monocytes antigens and found that the expression of this molecule account for about 85% of the total monocytes and are on CD8+ T cells was higher in cultures stimulated with characterized by the expression of IL-13Rα1, IL-10, and rSm29 compared with SLA alone. These results suggest that RANTES and by the expression of genes associated with anti- the CD8+ T cells became more activated in the presence apoptosis and wound healing properties. The intermediate of rSm29. There was, however, an increased expression of monocytes represent 5% of monocytes, express high levels CTLA-4 in CD4+ T cells, a molecule which is expressed of HLA-DR and are associated with antigen processing and to inhibit the T cell activation when rSmTSP-2 and PIII presentation to T cell and T cell activation. Nonclassical were added to the cultures. In vitro study has shown that subset are characterized by a low expression of CD14 and the addition of CTLA-4-Ig to block CD28-B7 interaction high expression of CD16 (CD14+CD16++). They represent in CL patients, PBMC cultures stimulated with Leishmania 10% of human monocytes and studies have shown that they antigen led to a downmodulation of IFN-γ, IL-10, and TNF express genes associated with cytoskeletal rearrangement secretion [27]. Our findings suggest that the CD4+ Tcells which account for their highly mobility and for the patrolling were downmodulated by the presence of the rSmTSP-2 and behavior in vivo [16, 47]. PIII. These antigens were also able to increase the frequency Since the roles of the different monocyte subsets in of the CD4+CD25highFoxp3+ regulatory T cells. cutaneous leishmaniasis remain unclear, we decided to In a survey performed by O’Neal et al. [51], the preva- evaluate not only their frequency in leishmaniasis patients, lence of S. mansoni infection in cutaneous leishmaniasis but also whether the addition of S. mansoni antigens would patients from the endemic area of Corte de Pedra, Bahia, alter the profile of these cells in an in vitro study. Brazil was 16.7% [51]. The authors also showed that 8 Journal of Parasitology Research coinfected individuals tended to have small lesion size, [3] O. Bacellar, H. Lessa, A. Schriefer et al., “Up-regulation of Th1- however in the univariated model, the presence of helminth type responses in mucosal leishmaniasis patients,” Infection coinfection was associated with delayed lesion healing. When and Immunity, vol. 70, no. 12, pp. 6734–6740, 2002. they evaluated infection with S. mansoni and Strongyloides [4] A. Ribeiro-de-Jesus, R. P. Almeida, H. Lessa, O. Bacellar, and stercoralis there was no difference in lesion healing compared E. M. Carvalho, “Cytokine profile and pathology in human to patients infected with geohelminths such as Ascaris leishmaniasis,” Brazilian Journal of Medical and Biological lumbricoides, Ancylostoma duodenale, and Trichuris trichiura. Research, vol. 31, no. 1, pp. 143–148, 1998. Studies conducted by the same group in the endemic [5] D. Sewell, Z. Qing, E. Reinke et al., “Immunomodulation of experimental autoimmune encephalomyelitis by helminth ova area of leishmaniasis in Brazil demonstrated that the use of immunization,” International Immunology, vol. 15, no. 1, pp. immunomodulatory agents such as granulocyte/macrophage 59–69, 2003. colony stimulating factor (GM-CSF) and pentoxifylline, an [6]A.Cooke,P.Tonks,F.M.Jonesetal.,“Infectionwith inhibitor of TNF production, can lead to faster healing time Schistosoma mansoni prevents insulin dependent diabetes and higher cure rate in cutaneous leishmaniasis [52–55]. It mellitus in non-obese diabetic mice,” Parasite Immunology, has also been reported that intralesional injections of GM- vol. 21, no. 4, pp. 169–176, 1999. CSF reduce healing time of CL ulcers by 50%. Moreover, [7] O. Atochina and D. Harn, “Prevention of psoriasis-like Santos et al. [53] showed that GM-CSF applied locally in lesions development in fsn/fsn mice by helminth glycans,” low doses as an adjuvant to pentavalent antimonial therapy, Experimental Dermatology, vol. 15, no. 6, pp. 461–468, 2006. significantly decreases the healing time of CL ulcers, reducing [8]A.M.Bafica,L.S.Cardoso,S.C.Oliveiraetal.,“Schistosoma the dose of antimony administered and/or the duration mansoniantigens alter the cytokine response in vitro during of antimonial therapy. These studies indicate that down- cutaneous leishmaniasis,” Memorias´ do Instituto Oswaldo modulation of the inflammatory response in CL patients is Cruz, vol. 106, pp. 856–863, 2011. associated with lesion cure and give support to the idea that [9]A.J.G.Simpson,P.Hagan,F.Hackett,P.OmerAli,andS.R. inhibition of the strong inflammatory response early after Smithers, “Epitopes expressed on very low M(r) Schistosoma mansoni adult tegumental antigens conform to a general Leishmania infection could avert tissue damage. pattern of life-cycle cross-reactivity,” Parasitology, vol. 100, In this context, the data presented herein suggest that S. part 1, pp. 73–81, 1990. mansoni antigens are capable of downregulating monocyte [10] F. C. Cardoso, J. M. R. Pinho, V. Azevedo, and S. C. Oliveira, and T lymphocyte response to the Leishmania antigen in “Identification of a new Schistosoma mansoni membrane- vitro, possibly through mechanisms that involve negative bound protein through bioinformatic analysis,” Genetics and signaling by CTLA-4 and the action of regulatory T cells. Molecular Research, vol. 5, no. 4, pp. 609–618, 2006. This knowledge could contribute to the development of [11] M. H. Tran, M. S. Pearson, J. M. Bethony et al., “Tetraspanins future strategies to prevent and treat cutaneous and mucosal on the surface of Schistosoma mansoni are protective antigens leishmaniasis. against schistosomiasis,” Nature Medicine,vol.12,no.7,pp. 835–840, 2006. [12] C. Hirsch and A. M. Goes, “Characterization of fractionated Conflict of Interests Schistosoma mansoni soluble adult worm antigens that elicit human cell proliferation and granuloma formation in vitro,” The authors have no conflict of interests concerning the work Parasitology, vol. 112, no. 6, pp. 529–535, 1996. reported in this paper. [13] C.Hirsch,C.S.Zouain,J.B.Alves,andA.M.Goes,“Induction of protective immunity and modulation of granulomatous Acknowledgments hypersensitivity in mice using PIII, an anionic fraction of Schistosoma mansoni adult worm,” Parasitology, vol. 115, part The authors would like to thank the people of the 1, pp. 21–28, 1997. leishmaniasis-endemic area in Corte de Pedra-Bahia for their [14] L. S. Cardoso, S. C. Oliveira, R. P. Souza et al., “Schistosoma participation in our study; Dr. Luiz Henrique Guimaraes,˜ mansoni antigens modulate allergic response in vitro in cells Dr. Paulo Machado, and Ednaldo Lago who assisted in data of asthmatic individuals,” Drug Development Research, vol. 72, collection and Hana Khidir and Whitney Oriana for the pp. 538–548, 2011. review of the paper. This work was supported by the Brazilian [15] L. Ziegler-Heitbrock, P. Ancuta, S. Crowe et al., “Nomencla- ture of monocytes and dendritic cells in blood,” Blood, vol. National Research Council (CNPq) and by the NIH Grant 116, no. 16, pp. e74–e80, 2010. no. NIH AI 088650. M. I. Araujo, A. M. Goes,´ S. O. Costa, [16] K.L.Wong,J.J.Tai,W.C.Wongetal.,“Geneexpressionprofil- and E. M. Carvalho are investigators supported by CNPq. ing reveals the defining features of the classical, intermediate, and non-classical human monocyte subsets,” Blood, vol. 118, References pp. 16–31, 2011. [17] J. Cros, N. Cagnard, K. Woollard et al., “Human CD14dim [1] P. Desjeux, “Leishmaniasis: public health aspects and control,” monocytes patrol and sense nucleic acids and viruses via TLR7 Clinics in Dermatology, vol. 14, no. 5, pp. 417–423, 1996. and TLR8 receptors,” Immunity, vol. 33, no. 3, pp. 375–386, [2] A.Schriefer,A.L.F.Schriefer,A.Goes-Neto´ et al., “Multiclonal 2010. Leishmania braziliensis population structure and its clinical [18] J. Y. Zhang, Z. S. Zou, A. Huang et al., “Hyper-activated pro- implication in a region of endemicity for American tegumen- inflammatory CD16+ monocytes correlate with the severity of tary leishmaniasis,” Infection and Immunity,vol.72,no.1,pp. liver injury and fibrosis in patients with chronic hepatitis B,” 508–514, 2004. PLoS ONE, vol. 6, no. 3, Article ID e17484, 2011. Journal of Parasitology Research 9

[19] Y. Rodriguez-Munoz, S. Martin-Vilchez, R. Lopez-Rodriguez [35] L. S. Cardoso, M. I. Araujo, A. M. Goes,´ L. G. Pac´ıfico, R. R. et al., “Peripheral blood monocyte subsets predict antiviral Oliveira, and S. C. Oliveira, “Polymyxin B as inhibitor of LPS response in chronic hepatitis C,” Alimentary Pharmacology & contamination of Schistosoma mansoni recombinant proteins Therapeutics, vol. 34, pp. 960–971, 2011. in human cytokine analysis,” Microbial Cell Factories, vol. 6, [20] J. Han, B. Wang, N. Han et al., “CD14high CD16+ rather than article 1, 2007. CD14lowCD16+ monocytes correlate with disease progression [36] M. S. Pearson, D. A. Pickering, H. J. McSorley et al., “Enhanced in chronic HIV-infected patients,” Journal of Acquired Immune protective efficacy of a chimeric form of the schistosomiasis Deficiency Syndromes, vol. 52, no. 5, pp. 553–559, 2009. vaccine antigen Sm-TSP-2,” PLOS Neglected Tropical Diseases, [21] E. L. Azeredo, P. C. Neves-Souza, A. R. Alvarenga et al., “Dif- vol. 6, article e1564, 2011. ferential regulation of toll-like receptor-2, toll-like receptor-4, [37] I. Follador, C. Araujo,´ O. Bacellar et al., “Epidemiologic and CD16 and human leucocyte antigen-DR on peripheral blood immunologic findings for the subclinical form of Leishmania monocytes during mild and severe dengue fever,” Immunology, braziliensis infection,” Clinical Infectious Diseases, vol. 34, no. vol. 130, no. 2, pp. 202–216, 2010. 11, pp. E54–E58, 2002. [22] G. Soares, A. Barral, J. M. Costa, M. Barral-Netto, and J. [38] L. R. V. Antonelli, W. O. Dutra, R. R. Oliveira et al., “Disparate Van Weyenbergh, “CD16+ monocytes in human cutaneous immunoregulatory potentials for double-negative (CD4− leishmaniasis: increased ex vivo levels and correlation with CD8−) αβ and γδ T cells from human patients with cutaneous clinical data,” Journal of Leukocyte Biology,vol.79,no.1,pp. leishmaniasis,” Infection and Immunity, vol. 74, no. 11, pp. 36–39, 2006. 6317–6323, 2006. [23] K. S. Hathcock, G. Laszlo, C. Pucillo, P. Linsley, and R. J. [39] S. T. Gaze, W. O. Dutra, M. Lessa et al., “Mucosal leishmaniasis Hodes, “Comparative analysis of B7-1 and B7-2 costimulatory patients display an activated inflammatory T-cell phenotype ligands: expression and function,” Journal of Experimental associated with a nonbalanced monocyte population,” Scandi- Medicine, vol. 180, no. 2, pp. 631–640, 1994. navian Journal of Immunology, vol. 63, no. 1, pp. 70–78, 2006. [24] R. R. Oliveira, K. J. Gollob, J. P. Figueiredo et al., “Schistosoma [40] P. N. Rocha, R. P. Almeida, O. Bacellar et al., “Down- mansoni infection alters co-stimulatory molecule expression regulation of Th1 type of response in early human American and cell activation in asthma,” Microbes and Infection, vol. 11, cutaneous leishmaniasis,” Journal of Infectious Diseases, vol. no. 2, pp. 223–229, 2009. 180, no. 5, pp. 1731–1734, 1999. [25] T. L. Walunas, D. J. Lenschow, C. Y. Bakker et al., “CTLA-4 can [41] M. I. A. S. Araujo, B. Hoppe, M. Medeiros et al., “Impaired function as a negative regulator of T cell activation,” Immunity, T helper 2 response to aeroallergen in helminth-infected vol. 1, no. 5, pp. 405–413, 1994. patiente with asthma,” Journal of Infectious Diseases, vol. 190, [26] J. F. Brunet, F. Denizot, and M. F. Luciani, “A new member no. 10, pp. 1797–1803, 2004. of the immunoglobulin superfamily—CTLA-4,” Nature, vol. [42] M. Hesse, C. A. Piccirillo, Y. Belkaid et al., “The pathogen- 328, no. 6127, pp. 267–270, 1987. esis of schistosomiasis is controlled by cooperating IL-10- [27] C. Favali, D. Costa, L. Afonso et al., “Role of costimulatory producing innate effector and regulatory T cells,” Journal of molecules in immune response of patients with cutaneous Immunology, vol. 172, no. 5, pp. 3157–3166, 2004. leishmaniasis,” Microbes and Infection, vol. 7, no. 1, pp. 86–92, [43] M. I. Araujo,´ B. S. Hoppe, M. Medeiros Jr., and E. M. 2005. Carvalho, “Schistosoma mansoni infection modulates the [28]C.I.Brodskyn,G.K.DeKrey,andR.G.Titus,“Influenceof immune response against allergic and auto-immune diseases,” costimulatory molecules on immune response to Leishmania Memorias do Instituto Oswaldo Cruz, vol. 99, no. 5, pp. 27–32, major by human cells in vitro,” Infection and Immunity, vol. 2004. 69, no. 2, pp. 665–672, 2001. [44] M. Rossol, S. Kraus, M. Pierer, C. Baerwald, and U. Wagner, [29] H. Yagi, T. Nomura, K. Nakamura et al., “Crucial role “The CD14bright CD16+ monocyte subset is expanded in of FOXP3 in the development and function of human rheumatoid arthritis and promotes expansion of the Th17 cell CD25+CD4+ regulatory T cells,” International Immunology, population,” Arthritis & Rheumatism, vol. 64, pp. 671–677, vol. 16, no. 11, pp. 1643–4656, 2004. 2011. [30] C. Baecher-Allan, J. A. Brown, G. J. Freeman, and D. A. Hafler, [45] W. K. Kim, Y. Sun, H. Do et al., “Monocyte heterogeneity “CD4+ CD25high regulatory cells in human peripheral blood,” underlying phenotypic changes in monocytes according to Journal of Immunology, vol. 167, no. 3, pp. 1245–1253, 2001. SIV disease stage,” Journal of Leukocyte Biology, vol. 87, no. 4, [31] C. Romagnani, M. Della Chiesa, S. Kohler et al., “Activation pp. 557–567, 2010. of human NK cells by plasmacytoid dendritic cells and its [46] L. Ziegler-Heitbrock, “The CD14+ CD16+ blood monocytes: modulation by CD4+ T helper cells and CD4+ CD25hi T their role in infection and inflammation,” Journal of Leukocyte regulatory cells,” European Journal of Immunology, vol. 35, no. Biology, vol. 81, no. 3, pp. 584–592, 2007. 8, pp. 2452–2458, 2005. [47]K.L.Wong,W.H.Yeap,J.J.Tai,S.M.Ong,T.M.Dang,andS. [32] Y. Belkaid and K. Tarbell, “Regulatory T cells in the con- C. Wong, “The three human monocyte subsets: implications trol of host-microorganism interactions,” Annual Review of for health and disease,” Immunology Research, vol. 53, pp. 41– Immunology, vol. 27, pp. 551–589, 2009. 57, 2012. [33] Y. Belkaid, C. A. Piccirillo, S. Mendez, E. M. Shevach, and D. [48] B. Passlick, D. Flieger, and H. W. Loms Ziegler-Heitbrock, L. Sacks, “CD4+CD25+ regulatory T cells control Leishmania “Identification and characterization of a novel monocyte major persistence and immunity,” Nature, vol. 420, no. 6915, subpopulation in human peripheral blood,” Blood, vol. 74, no. pp. 502–507, 2002. 7, pp. 2527–2534, 1989. [34] S. G. Reed, E. M. Carvalho, C. H. Sherbert et al., “In [49] L. S. Cardoso, S. C. Oliveira, and M. I. Araujo, “Schistosoma vitro responses to Leishmania antigens by lymphocytes from mansoniantigens as modulators of the allergic inflammatory patients with leishmaniasis or Chagas’ disease,” Journal of response in asthma,” Endocrine, Metabolic & Immune Disor- Clinical Investigation, vol. 85, no. 3, pp. 690–696, 1990. ders, vol. 12, pp. 24–32, 2012. 10 Journal of Parasitology Research

[50] L. G. G. Pac´ıﬁco, F. A. V.Marinho, C. T. Fonseca et al., “Schisto- soma mansoni antigens modulate experimental allergic asthma in a murine model: a major role for CD4+ CD25+ Foxp3+ T cells independent of interleukin-10,” Infection and Immunity, vol. 77, no. 1, pp. 98–107, 2009. [51] S. E. O’Neal, L. H. Guimaraes,˜ P. R. Machado et al., “Inﬂuence of helminth infections on the clinical course of and immune response to Leishmania braziliensis cutaneous leishmaniasis,” Journal of Infectious Diseases, vol. 195, no. 1, pp. 142–148, 2007. [52] H. A. Lessa, P. Machado, F. Lima et al., “Successful treatment of refractory mucosal leishmaniasis with pentoxifylline plus antimony,” American Journal of Tropical Medicine and Hygiene, vol. 65, no. 2, pp. 87–89, 2001. [53] J. B. Santos, A. R. De Jesus, P. R. Machado et al., “Antimony plus recombinant human granulocyte-macrophage colony- stimulating factor applied topically in low doses enhances healing of cutaneous leishmaniasis ulcers: a randomized, double-blind, placebo-controlled study,” Journal of Infectious Diseases, vol. 190, no. 10, pp. 1793–1796, 2004. [54]R.P.Almeida,J.Brito,P.L.Machadoetal.,“Successful treatment of refractory cutaneous leishmaniasis with GM-CSF and antimonials,” American Journal of Tropical Medicine and Hygiene, vol. 73, no. 1, pp. 79–81, 2005. [55] R. Almeida, A. D’Oliveira Jr., P. Machado et al., “Randomized, double-blind study of stibogluconate plus human granulocyte macrophage colony-stimulating factor versus stibogluconate alone in the treatment of cutaneous leishmaniasis,” Journal of Infectious Diseases, vol. 180, no. 5, pp. 1735–1737, 1999. Hindawi Publishing Corporation Journal of Parasitology Research Volume 2012, Article ID 480824, 7 pages doi:10.1155/2012/480824

Research Article Transcriptional Proﬁle and Structural Conservation of SUMO-Speciﬁc Proteases in Schistosoma mansoni

Roberta Verciano Pereira,1 Fernanda Janku Cabral,2 MatheusdeSouzaGomes,3 Liana Konovaloff Jannotti-Passos,4 William Castro-Borges,1 and Renata Guerra-Sa´ 1

1 Departamento de Ciências Biologicas/N´ ucleo´ de Pesquisas em Ciências Biologicas,´ Instituto de Ciências Exatas e Biologicas,´ Universidade Federal de Ouro Preto, Campus Morro do Cruzeiro, 35400-000 Ouro Preto, MG, Brazil 2 Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de Sao˜ Paulo, Av. Lineu Prestes, 1374-Butantan, 05508-900 Sao˜ Paulo, SP, Brazil 3 Instituto de Genética e Bioqu´ımica, Universidade Federal de Uberlandia,ˆ Av. Getulio´ Vargas, Palacio´ dos Cristais-Centro, 38700-126 Patos de Minas, MG, Brazil 4 Fundaçao˜ Oswaldo Cruz, Centro de Pesquisas René Rachou, Laboratorio´ de Malacologia, Av. Augusto de Lima, 1715-Barro Preto, 30190-002 Belo Horizonte, MG, Brazil

Correspondence should be addressed to Renata Guerra-Sa,´ [email protected]

Received 24 April 2012; Revised 9 August 2012; Accepted 23 August 2012

Academic Editor: Cristina Toscano Fonseca

Copyright © 2012 Roberta Verciano Pereira et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Small ubiquitin-related modifier (SUMO) is involved in numerous cellular processes including protein localization, transcription, and cell cycle control. SUMOylation is a dynamic process, catalyzed by three SUMO-specific enzymes and reversed by Sentrin/SUMO-specific proteases (SENPs). Here we report the characterization of these proteases in Schistosoma mansoni. Using in silico analysis, we identified two SENPs sequences, orthologs of mammalian SENP1 and SENP7, confirming their identities and conservation through phylogenetic analysis. In addition, the transcript levels of Smsenp1/7 in cercariae, adult worms, and in vitro cultivated schistosomula were measured by qRT-PCR. Our data revealed upregulation of the Smsenp1/7 transcripts in cercariae and early schistosomula, followed by a marked differential gene expression in the other analyzed stages. However, no significant difference in expression profile between the paralogs was observed for the analyzed stages. Furthermore, in order to detect deSUMOylating capabilities in crude parasite extracts, SmSENP1 enzymatic activity was evaluated using SUMO-1- AMC substrate. The endopeptidase activity related to SUMO-1 precursor processing did not differ significantly between cercariae and adult worms. Taken together, these results support the developmentally regulated expression of SUMO-specific proteases in S. mansoni.

1. Introduction to ubiquitylation. The sequential actions of E1, E2, and E3 enzymes catalyze the attachment of SUMO to target pro- Reversible posttranslational modification by ubiquitin and teins, while deconjugation is promoted by SUMO-specific ubiquitin-like proteins (Ubls) plays a crucial role in dynamic proteases. In addition, like other Ubls, SUMO polypeptide regulation of protein function, fate, binding partners, is synthesized as an inactive precursor, which requires a C- activity, and localization. Small ubiquitin-related modifier terminal cleavage to expose the glycine residue for substrate (SUMO) is a member of Ubls family that is covalently conjugation. This process is also catalyzed by SUMO-specific attached to lysine residues of their protein targets in protease through its hydrolase activity. The catalytic activity cells, altering function and subcellular localization [1]. is maintained within a highly conserved 200 amino acid SUMOylation has been implicated in the regulation of region at the C-terminus of the proteases [4–6]. numerous biological processes including transcription [2] Recently, the importance of reversible SUMOylation for andcellcyclecontrol[3]. SUMO conjugation mechanism normal cell physiology has been demonstrated. Excessive is a highly dynamic and reversible process closely related SUMO conjugation using knockouts for either SENP1 or 2 Journal of Parasitology Research

SENP2 induced an embryonic lethal phenotype [7, 8]and CO2. Then the recovered schistosomula were washed with deregulation of either SUMO conjugation or deconjugation RPMI 1640 until no tails were detected. For subsequent can contribute to cancer progression as reviewed by [9]. In incubations, the parasites were maintained in M169 medium humans there are six SENP enzymes (SENP1, -2, -3, -5, supplemented with 10% FBS, penicillin, and streptomycin at -6, and -7) that deconjugate mono-SUMOylated proteins 100 μg/mL and 5% Schneider’s medium [13]at37◦Con5% or disassemble polymeric SUMO side chains, while in CO2 during3.5h,1,2,3,5,and7days. Saccharomyces cerevisiae there are only two deSUMOylating enzymes, Ulp1 and Ulp2. Based on these activities and 2.3. Identification and Computational Analysis of affinities for SUMO isoforms, SENPs can be categorized into SmSENP1/7.SmSENP1/7 sequences were retrieved from three independent subfamilies: SENP1 and SENP2; SENP3 the S. mansoni genome database version 5.0 available and SENP5; SENP6 and SENP7 [9]. at http://www.genedb.org/genedb/smansoni/ through The initial molecular characterization of SUMOylation BLAST searches. Amino acid sequences from Drosophila pathway in S. mansoni showed the presence of two SUMO melanogaster, Caenorhabditis elegans and Homo sapiens paralogs called SMT3C and SMT3B [10], suggesting a variety SENP orthologs were used as queries. The BLASTp of targeted substrates for these modifications. Recently, our algorithm, underpinned by the Pfam (v26.0), allowed group reported the conservation of SUMO conjugating detection of conserved protein domains or motifs from S. enzyme SmUBC9 by phylogeny, primary and modeled ter- mansoni sequences. Multiple alignments of SmSENP1/7 tiary structures, as well as mRNA transcription and protein were performed by ClustalX 2.0 and phylogenetic analysis levels throughout the S. mansoni life cycle. Our data showed was conducted in MEGA 5 [14]. A phylogenetic tree of that Smubc9 transcription levels are upregulated in early these sequences was inferred using the Neighbor-Joining schistosomula, suggesting the utilization of this posttrans- method [15]. The bootstrap consensus tree inferred from lational modification mechanism S. mansoni development, 1000 replicates was used to represent the evolutionary particularly at the initial phase of invasion of the vertebrate history of the taxa analyzed. Branches corresponding to host [11]. partitions reproduced in less than 50% bootstrap replicates To extend the investigation of the SUMOylation pathway are collapsed. The percentage of replicate trees in which the in S. mansoni, we firstly retrieved through homology-based associated taxa clustered together in the bootstrap test (1000 searches putative SmSENPs using the publically available S. replicates) is shown next to the branches. The tree was drawn mansoni databases. Furthermore, the levels of Smsenp1/7 to scale, with branch lengths in the same units as those of transcripts were evaluated by qRT-PCR using mRNA from the evolutionary distances used to infer the phylogenetic cercariae, adult worms and mechanically-transformed schis- tree. All positions containing gaps and missing data were tosomula (MTS) in vitro-cultivated during 3.5 h, 1, 2, 3, 5, eliminated from the dataset. and 7 days. We also measured the endopeptidase activities of SmSENPs in cercariae and adult worm crude extracts. The present study reveals differences in the gene expression 2.4. RNA Preparation and Expression Analysis of Smsenp1/7 profile of Smsenp1/7 during schistosomula development. by qRT-PCR. Total RNA from adult worms, cercariae, and In addition, similar SmSENP1 activity was observed in schistosomula was obtained using a combination of the cercariae and adult worms, suggesting the importance of this Trizol reagent (GIBCO-BRL) and chloroform for extraction, proteolytic activity during the parasite’s life cycle. and then on column purified using the “SV total RNA Isolation System” (Promega, Belo Horizonte, Brazil). The preparation was treated with RNase-free DNase I in 3 2. Materials and Methods different rounds by decreasing enzyme concentration (RQ1 DNase; Promega). RNA was quantified using a spectropho- 2.1. Ethics Statement. All experiments involving animals tometer and an aliquot containing 1 μgoftotalRNAreverse were authorized by the Ethical Committee for Animal Care transcribed using an oligodT primer from the Thermoscript of Federal University of Ouro Preto, and were in accordance RT-PCR System (Invitrogen Sao˜ Paulo, Brazil) as described with the national and international regulation accepted for by the manufacturer. The efficiency of DNAse I treatment laboratory animal use and care. was evaluated by PCR amplification of the cDNA reaction mix without the addition of the Thermoscript enzyme. S. 2.2. Parasites. S. mansoni. LE strain was maintained by mansoni specific SENP1/7 primers were designed using the routine passage through Biomphalaria glabrata snails and program GeneRunner for SmSENP1 (GeneDB access num- BALB/c mice. The infected snails were induced to shed ber Smp 033260.2) (forward 5-AGGAAACGGAGGCGG- cercariae under light exposure for 2 h and the cercariae were GATTC-3, reverse 5-ACACTGGAGACACGGGATGAGC- recovered by sedimentation on ice. Adult worm parasites 3)andforSmSENP7 (GeneDB access number Smp 159120) were obtained by liver perfusion of mice after 50 days of (forward 5-TCAGTTACACGGCCCTTTATC-3, reverse 5- infection. Mechanically transformed schistosomula (MTS) CCTGAGAAGTGGATGCGATC-3). Reverse-transcribed were prepared as described by Harrop and Wilson [12]. cDNA samples were used as templates for PCR amplification Briefly, cercariae were recovered, and washed in RPMI 1640 using SYBR Green Master Mix UDG-ROX (Invitrogen) medium (Invitrogen), before vortexing at maximum speed and 7300 Real Time PCR System (Applied Biosystems, for 90 s and immediately cultured during 3.5 h at 37◦C, 5% Rio de Janeiro, Brazil). Specific primers for S. mansoni Journal of Parasitology Research 3

α-tubulin were used as an endogenous control (GenBank NP 065705.3). Alignment of their catalytic domains with the access number M80214) (forward 5-CGTATTCGCAAG- human SENPs showed the presence of the three essential TTGGCTGACCA-3, reverse 5-CCATCGAAGCGCAGT- catalytic residues (Cys-His-Asp), highlighted in Figure 2. GATGCA-3)[16]. The efficiency of each pair of primers was evaluated according to the protocol developed by the Applied 3.2. Smsenp1/7 Are Differentially Expressed in S. mansoni. Biosystems application (cDNA dilutions were 1 : 10, 1 : 100 The gene expression profile of Smsenp1 and Smsenp7 and 1 : 1000). For all investigated transcripts three biological was determined using qRT-PCR. We designed specific replicates were performed and their gene expression normal- primers to amplify Smsenp1 and Smsenp7 transcripts in α −ΔCt ized against the -tubulin transcript according to the 2 the cercariae-schistosomula transition and adult worm method [17] using the Applied Biosystems 7300 software. (Figure 3). We observed that Smsenp1 and Smsenp7 transcripts are expressed in basal levels from MTS-2d to 7 days 2.5. Endopeptidase Activity. To determine the enzymatic and adult worms. The levels of the SENPs transcripts were activity of SENP proteases present in adult worms and significantly higher in MTS-3.5 h being approximately 10- cercariae crude extracts we used the fluorogenic substrate fold higher when compared to levels in adult worms and SUMO-1-AMC that is specific for SENP1 (Enzo Life Sci- during schistosomula development (MTS-2, 3, 5, and 7 days) ences, Sao˜ Paulo, Brazil). In these assays 10 μgoftotal and 3-fold higher when compared to cercariae and MTS-1d. protein were used and 0,45 μM of the fluorogenic substrate in We also observed that Smsenp1 and Smsenp7 transcription 50 mM Tris-HCl pH 8, 10 mM MgCl2± 0,45 μMSUMO-1- levels were not significantly different (P < 0.05) within a aldehyde (Enzo Life Sciences) to control for specific enzyme given stage, except for MTS-3.5 h where Smsenp7 levels are inhibition. Each enzymatic assay was conducted in a final 3 fold-higher than Smsenp1. volume of 100 μL for both extracts, followed by 60 minutes ◦ incubation at 37 C. The reaction was stopped by addition of 3.3. SmSENP1 Enzymatic Activity in Cercariae and Adult 2 mL of 99.5% ethanol. The fluorometric readings were taken Worms. In vitro endopeptidase assays were performed using at wavelengths of 380 nm (excitation) and 440 nm (emission) crude extract from cercariae and adult worms (Figure 4). The in a spectrofluorimeter (Turner QuantechTM Fluorometer), μ SmSENP1 enzymatic activity was slightly higher in cercariae and the results expressed in fluorescence units per goftotal compared to adult worms, although the levels were not protein. significantly different (P < 0.05). Enzyme activities were also recorded in the presence of a commercially available 2.6. Statistical Analysis. Statistical analysis was performed HsSENP1 inhibitor but significant differences were not using GraphPad Prism version 5.0 software package (Irvine, observed in the analyzed stages. CA, USA). Normality of the data was established using one way analysis of variance (ANOVA). Tukey post tests were used to investigate significant differential expression 4. Discussion of SUMO proteases throughout the investigated stages. In Previous reports from our group showed the presence of ff all cases, the di erences were considered significant when P two SUMO paralogs (SMT3C and SMT3B) in S. mansoni, < values were 0.05. exhibiting differential expression between larvae and adult worms. The electrophoretic pattern of SUMO-conjugated 3. Results molecules also differed comparing the analyzed stages [10]. Furthermore, our group demonstrated a differential expres- 3.1. SUMO Specific-Proteases: The Conservation of sion profile for UBC9 throughout the parasite life cycle [11]. SmSENP1/7. In silico analysis of SmSENPs revealed the The structural SUMO conservation in S. mansoni reinforces conserved putative domains of SUMO proteases in S. SUMO as a candidate for a transcription regulator and a mansoni. The parasite database has two sequences encoding degradation signal by facilitating ubiquitylation of certain putative SENPs: Smp 033260.2 (putative SENP1) and target proteins during parasite development. Smp 159120 (putative SENP7). SENP1 has an alternative In order to understand the role of SUMOylation in S. spliced form annotated as Smp 033260.1. We evaluated mansoni, we evaluated the conservation of SUMO proteases the conservation of these proteins throughout evolution and their gene expression profile in cercariae, schistosomula, using a phylogenetic approach. Our results showed that and adult worm. Firstly, we retrieved, through homology- each SENP was grouped in 2 distinct clades, reinforcing based searches, two sequences containing conserved domains their structural conservation among the thirteen analyzed of SUMO proteases. A phylogenetic approach then revealed orthologs (Figure 1). two putative proteases closely associated to human SENP1 To confirm that these predicted proteases are cysteine- and SENP7. Alignment of parasite sequences with their family members, analyses using the Pfam protein domain human counterparts also demonstrated conservation of the database were conducted. For both SmSENPs entries a catalytic triad for both proteases [18]. The active site residues conserved Peptidase C48 domain was revealed. Our in silico within the core domain of Ulp1 and Ulp2 have been proven analysis demonstrated SmSENP1 more closely related to to be necessary for catalytic activity and in vivo function in HsSENP1 (GenBank access number NP 055369.1), whereas yeast [19, 20]. Furthermore, Ulp1 lacks obvious sequence SmSENP7 is related to HsSENP7 (GenBank access number similarity to any known deubiquitinating enzyme so it is 4 Journal of Parasitology Research

NP 001254524.1 (Homo sapiens) 99 94 NP 659100.1 (Mus musculus)

XP 001343517.1 (Danio rerio)

XP 002596928.1 (Branchiostoma ﬂoridae)

XP 797423.3 (Strongylocentrotus purpuratus)

XP 003251079.1 (Apis mellifera) SENP1 or ULP1-like NP 573362.1 (Drosophila melanogaster)

Smp 033260.2 94 AAX27951.2 (Schistosoma japonicum)

XP 002648991.1 (Caenorhabditis briggsae)

85 NP 498095.3 (Caenorhabditis elegans)

NP 191978.3 (Arabidopsis thaliana)

NP 015305.1 (Saccharomyces cerevisiae S288c)

NP 195088.2 (Arabidopsis thaliana)

NP 012233.1 (Saccharomyces cerevisiae S288c)

XP 003727099.1 (Strongylocentrotus purpuratus)

XP 002630625.1 (Caenorhabditis briggsae) 83 NP 494914.1 (Caenorhabditis elegans)

XP 002590318.1 (Branchiostoma ﬂoridae) SENP7 or XP 001122456.2 (Apis mellifera) 38 ULP2-like 31 NP 572827.1 (Drosophila melanogaster)

Smp 159120 86 CAX72652.1 (Schistosoma japonicum)

XP 003201158.1 (Danio rerio)

NP 001070671.1 (Homo sapiens) 55 78 NP 001003971.1 (Mus musculus)

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 Figure 1: Consensus phylogenetic tree based on amino acid sequences of SmSENPs. The tree construction and analysis of bootstrap were performed using ClustalX 2.0 and MEGA 4.0. For the consensus tree and reliability of the branches formed test was used phylogenetic bootstrap using 1000 replicates for each sequence, and 50% the minimum for considering the branch reliably. Organisms: Hs (Homo sapiens), Mm (Mus musculus), Rn (Rattus norvegicus), Xl (Xenopus laevis), Sm (Schistosoma mansoni), Sj (Schistosoma japonicum), Dm (Drosophila melanogaster), Cb (Caenorhabditis briggsae) and Ce (Caenorhabditis elegans). Journal of Parasitology Research 5

::::∗∗∗∗∗∗∗ ∗ ∗∗∗∗∗∗∗∗ :∗∗∗ ∗∗∗∗∗∗ :.:∗ ∗ Hs SENP7 VPIHLG-VHWCLAVVD-----ITYYDSMG------IPQQMNGSDCGMFACKY Sm SENP7 IPIHDRGMHWCLSCID-----ITYYDSMG------VPQQYNGSDCGVFLCTF :∗ :∗∗∗ .:∗∗ : .::∗ ∗∗∗ : ::∗∗∗∗∗ .∗∗ .∗∗∗ :∗∗∗ :∗ Hs SENP1 VPVNESSHWYLAVIC------RPCILILDSLKA------VPKQDNSSDCGVYLLQY Sm SENP1 IPINECAHWFLGLVC------MPCVLLFDSLPC------VPVQSNLVDCGIYLLHY

Figure 2: S. mansoni has two predicted SUMO-Speciﬁc Proteases. ClustalX 2.0 alignment of SENP1 and SENP7 catalytic residues from human and S. mansoni. The domains position of proteins in S. mansoni were based on Pfam database as well as the e-value score. The grey boxes represent the conserved domains based on Pfam database. Aligned catalytic residues are denoted by the black box. ∗, :, and . indicate, respectively, identical residues, highly conserved amino acid substitution, and conserved amino acid substitution.

90 ∗∗ ∗ 75 60

45 ∗∗ ∗

30 ∗ ∗∗∗ 15 ∗∗ ∗∗∗

5 ∗∗∗ ∗∗∗ ∗∗∗ ∗∗ ∗∗∗ 4 ∗∗∗ ∗∗ ∗∗∗ 3 ∗∗ ∗∗ Relative expression of expression SmSENPs Relative ∗∗∗ 2 ∗∗∗ 1 0 MTS-1 d MTS-1 d MTS-2 d MTS-3 d MTS-5 d MTS-7 Cercariae MTS-3, 5 h 5 MTS-3, Adult worm Adult SENP1 SENP7 Figure 3: SmSENPs are differentially expressed throughout the S. mansoni life cycle. The mRNA expression levels were measured based on three replicates, for each of the following stages: adult worms, cercariae, MTS-3.5 h, 1, 2, 3, 5, and 7 days using quantitative RT- PCR. Expression levels were calibrated according to the comparative 2−ΔCt method, using the constitutively expressed Smα-tubulin as an endogenous control (one-way variance analysis followed by Tukey pairwise comparison P < 0.05). ∗Different from adult worm, ∗∗different from cercariae, and ∗∗∗different from MTS-3.5 h.

30 unlikely that it is able to process ubiquitin-linked substrates [21]. Considering that the SUMOylation pathway dictates the function of a variety of substrates [22], and the existence 20 ∗ of two SUMO paralogs in S. mansoni [10], it is plausible g/min) µ that the worm utilizes distinct SUMO proteases for SUMO maturation and deSUMOylation of target substrates. Dis- crimination between SUMO-1 and SUMO-2/3 paralogs may 10 also account for distinct functions of SENP proteases in

Fluorescence ( Fluorescence S. mansoni. In mammalian cells, proteomic studies have identified proteins that are selectively modified by either 0 SUMO-1 or SUMO-2/3 [23]. It is well established that Cercariae Adult worms SENP1 processes SUMO-1 in preference to SUMO-2, and has a very low catalytic constant for SUMO-3 [24]. In SUMO1-AMC contrast, the recent characterization of the catalytic domain SUMO1 aldehyde of SENP7 in humans demonstrated a greater deconjugating Figure 4: SmSENP1 enzymatic activity in S. mansoni. The fluores- activity for SUMO-2/3 chains [25]. As discussed by Shen [26] cence levels were measured based on three replicates, in cercariae SENP7 acts as a SUMO-2/3 specific protease that is likely and adult worm stages. Statistical analysis was performed using t to regulate the metabolism of polySUMO-2/3 rather than tests followed by unpaired test P < 0.05. ∗Different from cercariae. SUMO-1 conjugation in vivo. 6 Journal of Parasitology Research

In the present investigation, the transcriptional profile [3] H. D. Ulrich, “Regulating post-translational modifications of of SUMO proteases was also evaluated by qPCR, using theeukaryoticreplicationclampPCNA,”DNA Repair, vol. 8, total RNA from distinct stages of the S. mansoni life no. 4, pp. 461–469, 2009. cycle. The gene expression data revealed similar expression [4] D. Mukhopadhyay and M. Dasso, “Modification in reverse: the profile for Smsenp1 and Smsenp7 at any given stage, but SUMO proteases,” Trends in Biochemical Sciences, vol. 32, no. with a remarkable differential expression for both proteases 6, pp. 286–295, 2007. [5] J. H. Kim and S. H. Baek, “Emerging roles of desumoylating comparing the larvae and adult stages. The differential gene enzymes,” Biochimica et Biophysica Acta, vol. 1792, no. 3, pp. expression profile observed for Smsenp1/7 throughout the S. 155–162, 2009. mansoni life cycle attests for the distinct patterns of SUMO [6] Z. Xu, H. Y. Chan, W. L. Lam et al., “SUMO proteases: conjugates observed during parasite development [10]. Poly- redox regulation and biological consequences,” Antioxidants SUMOylation has been reported to increase during heat- and Redox Signaling, vol. 11, no. 6, pp. 1453–1484, 2009. shock treatment and stress conditions, with SENP7 being [7]J.Cheng,X.Kang,S.Zhang,andE.T.H.Yeh,“SUMO- responsible for dismantling SUMO polymers [25, 27]. Given specific protease 1 is essential for stabilization of HIF1α during that the levels of Smsenp1/7 transcripts were significantly hypoxia,” Cell, vol. 131, no. 3, pp. 584–595, 2007. higher in cercariae and MTS-3.5 h, we suggest a stress- [8] S. Y. Chiu, N. Asai, F. Costantini, and W. Hsu, “SUMO- induced expression of the SUMOylation machinery during specific protease 2 is essential for modulating p53-Mdm2 in the parasite’s body adaptation to the new metabolic and development of trophoblast stem cell niches and lineages,” morphological alterations it undergoes [28]. Our findings are PLOS Biology, vol. 6, no. 12, p. e310, 2008. [9] T. Bawa-Khalfe and E. T. H. Yeh, “SUMO losing balance: also in agreement with Pereira [11] which demonstrated a SUMO proteases disrupt SUMO homeostasis to facilitate similar pattern of gene expression profile for Smubc9 in the cancer development and progression,” Genes and Cancer, vol. aforementioned parasite stages. 1, no. 7, pp. 748–752, 2010. We have not observed a positive correlation between [10] F. J. Cabral, O. S. Pereira Jr., C. S. Silva, R. Guerra-Sa,´ differential transcriptional levels of Smsenp1 and enzymatic andV.Rodrigues,“Schistosoma mansoni encodes SMT3B activity evaluated using synthetic SUMO-1-AMC substrate. and SMT3C molecules responsible for post-translational The levels of enzymatic activity for SmSENP1 were slightly modification of cellular proteins,” Parasitology International, higher in extracts of cercariae compared to adult worms. In vol. 57, no. 2, pp. 172–178, 2008. the presence of a specific inhibitor, no significant change in [11] R. V. Pereira, F. J. Cabral, M. S. Gomes et al., “Molecular enzymatic activity was detected. This may account for the characterization of SUMO E2 conjugation enzyme: differen- limited amino acid sequence identity (28%) shared between tial expression profile in Schistosoma mansoni,” Parasitology SmSENP1 and its human ortholog, from which the synthesis Research, vol. 109, no. 6, pp. 1537–1546, 2011. of SUMO-1 aldehyde is based. Additional studies aimed at [12] R. Harrop and R. A. Wilson, “Protein synthesis and release by elucidating substrate selection of SmSENP1/7 in S. mansoni cultured schistosomula of Schistosoma mansoni,” Parasitology, vol. 107, no. 3, pp. 265–274, 1993. and a detailed kinetic analysis of their enzymatic activities, [13] P. F. Basch and J. J. DiConza, “In vitro development of particularly during the cercarie to schistosomula transition Schistosoma mansoni cercariae,” Journal of Parasitology, vol. 63, may provide further insights into the biological activities no. 2, pp. 245–249, 1977. of these enzymes and the role of SUMO conjugation in S. [14] S. Kumar, K. Tamura, and M. Nei, “MEGA3: integrated mansoni. software for Molecular Evolutionary Genetics Analysis and sequence alignment,” Briefings in bioinformatics, vol. 5, no. 2, Acknowledgments pp. 150–163, 2004. [15] N. Saitou and M. Nei, “The neighbor-joining method: a The authors thank the transcriptome initiatives: Sao˜ Paulo new method for reconstructing phylogenetic trees,” Molecular Transcriptome Consortium; Minas Gerais GenomeNetwork; Biology and Evolution, vol. 4, no. 4, pp. 406–425, 1987. Wellcome Trust Genome Iniatiative (UK). This work was [16] P. J. Webster, K. A. Seta, S. C. Chung, and T. E. Mansour, α supported by the following Brazilian research agencies: “A cDNA encoding an -tubulin from Schistosoma mansoni,” Molecular and Biochemical Parasitology, vol. 51, no. 1, pp. 169– FAPEMIG (Fundaçao˜ de Amparo a` Pesquisa do Estado de 170, 1992. Minas Gerais, CBB 0558/09), Nucleo´ de Bioinformatica´ da [17] K. J. Livak and T. D. Schmittgen, “Analysis of relative gene Universidade Federal de Ouro Preto (NuBio UFOP) and expression data using real-time quantitative PCR and the 2- CNPq (Conselho Nacional de Desenvolvimento Cient´ıfico e ΔΔCT method,” Methods, vol. 25, no. 4, pp. 402–408, 2001. Tecnologico).´ [18] S. J. Li and M. Hochstrasser, “The Ulp1 SUMO isopeptidase: distinct domains required for viability, nuclear envelope References localization, and substrate specificity,” Journal of Cell Biology, vol. 160, no. 7, pp. 1069–1081, 2003. [1] J. R. Gareau and C. D. Lima, “The SUMO pathway: emerging [19] S. J. Li and M. Hochstrasser, “A new protease required for cell- mechanisms that shape specificity, conjugation and recogni- cycle progression in yeast,” Nature, vol. 398, no. 6724, pp. 246– tion,” Nature Reviews Molecular Cell Biology, vol. 11, no. 12, 251, 1999. pp. 861–871, 2010. [20] A. V. Strunnikov, L. Aravind, and E. V. Koonin, “Saccha- [2] J. M. P. Desterro, M. S. Rodriguez, and R. T. Hay, “SUMO- romyces cerevisiae SMT4 encodes an evolutionarily conserved 1 modification of IκBα inhibits NF-κB activation,” Molecular protease with a role in chromosome condensation regulation,” Cell, vol. 2, no. 2, pp. 233–239, 1998. Genetics, vol. 158, no. 1, pp. 95–107, 2001. Journal of Parasitology Research 7

[21] S. J. Li and M. Hochstrasser, “The yeast ULP2 (SMT4) gene encodes a novel protease specific for the ubiquitin-like Smt3 protein,” Molecular and Cellular Biology,vol.20,no.7,pp. 2367–2377, 2000. [22] G. Rosas-Acosta, W. K. Russell, A. Deyrieux, D. H. Russell, and V. G. Wilson, “A universal stategy for proteomic studies of SUMO and other ubiquitin-like modifiers,” Molecular and Cellular Proteomics, vol. 4, no. 1, pp. 56–72, 2005. [23] A. C. O. Vertegaal, J. S. Andersen, S. C. Ogg, R. T. Hay, M. Mann, and A. I. Lamond, “Distinct and overlapping sets of SUMO-1 and SUMO-2 target proteins revealed by quantitative proteomics,” Molecular and Cellular Proteomics, vol. 5, no. 12, pp. 2298–2310, 2006. [24] L. N. Shen, C. Dong, H. Liu, J. H. Naismith, and R. T. Hay, “The structure of SENP1-SUMO-2 complex suggests a structural basis for discrimination between SUMO paralogues during processing,” Biochemical Journal, vol. 397, no. 2, pp. 279–288, 2006. [25] C. D. Lima and D. Reverter, “Structure of the human SENP7 catalytic domain and poly-SUMO deconjugation activities for SENP6 and SENP7,” Journal of Biological Chemistry, vol. 283, no. 46, pp. 32045–32055, 2008. [26] L. N. Shen, M. C. Geoffroy, E. G. Jaffray,andR.T.Hay, “Characterization of SENP7, a SUMO-2/3-specific isopeptidase,” Biochemical Journal, vol. 421, no. 2, pp. 223–230, 2009. [27] F. Golebiowski, I. Matic, M. H. Tatham et al., “System-wide changes to sumo modifications in response to heat shock,” Science Signaling, vol. 2, no. 72, p. ra24, 2009. [28] J. R. Lawson and R. A. Wilson, “Metabolic changes associated with the migration of the schistosomulum of Schistosoma mansoni in the mammal host,” Parasitology, vol. 81, no. 2, pp. 325–336, 1980. Hindawi Publishing Corporation Journal of Parasitology Research Volume 2012, Article ID 541268, 8 pages doi:10.1155/2012/541268

Review Article Schistosoma Tegument Proteins in Vaccine and Diagnosis Development: An Update

Cristina Toscano Fonseca,1, 2 Gardeniaˆ Braz Figueiredo Carvalho,1 Clarice Carvalho Alves,1 and Tatiane Teixeira de Melo1

1 Laboratorio´ de Esquistossomose, Centro de Pesquisas René Rachou, Fundaçao˜ Oswaldo Cruz, Avenida Augusto de Lima 1715, Belo Horizonte,MG 30190-002, Brazil 2 Instituto Nacional de Ciências e Tecnologia em Doenças Tropicais (INCT-DT), Avenida Augusto de Lima 1715, Belo Horizonte, MG 30190-002, Brazil

Correspondence should be addressed to Cristina Toscano Fonseca, ctoscano@cpqrr.ﬁocruz.br

Received 27 July 2012; Accepted 24 September 2012

Academic Editor: Andrea Teixeira-Carvalho

Copyright © 2012 Cristina Toscano Fonseca et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The development of a vaccine against schistosomiasis and also the availability of a more sensitive diagnosis test are important tools to help chemotherapy in controlling disease transmission. Bioinformatics tools, together with the access to parasite genome, published recently, should help generate new knowledge on parasite biology and search for new vaccines or therapeutic targets and antigens to be used in the disease diagnosis. Parasite surface proteins, especially those expressed in schistosomula tegument, represent interesting targets to be used in vaccine formulations and in the diagnosis of early infections, since the tegument represents the interface between host and parasite and its molecules are responsible for essential functions to parasite survival. In this paper we will present the advances in the development of vaccines and diagnosis tests achieved with the use of the information from schistosome genome focused on parasite tegument as a source for antigens.

1. Introduction a combination of traditional Sanger capillary sequencing and deep-coverage Illumina sequencing that refined gene pre- Schistosomiasis is still a significant public health problem diction resulting in a reduction in the number of predicted ff in tropical countries despite the existence of e ective drugs genes from 11.809 to 10.852. Illumina-based technology was against the parasite [1]. Chemotherapy as a strategy for dis- also used in Schistosoma haematobium genome sequencing, ff ease control has proved ine ective in controlling transmis- which described 13.073 genes [6]. sion [1] therefore, the development of a vaccine against the Simultaneously to genome publication, an important disease and also a more sensitive diagnosis test is necessary to tool to access and analyze parasite genome has been devel- assist chemotherapy in control programs [1, 2]. oped, the SchistoDB (http://www.schistodb.net/) database In this context, the recent availability of schistosome [7]. The SchistoDB enables access to information on the genomes information represents an important toll to be parasite genome even to those researchers not specialized in used in the discovery of new targets for vaccine and diag- computer language. The current 3.0 database version pro- nosis. Schistosoma mansoni genome, published in 2009 [3] vides access to the latest draft of S. mansoni genome sequence described 11.809 genes while Schistosoma japonicum genome and annotation and also to S. japonicum and S. haematobium [4] has been described to be composed of 13.469 genes. genome annotation. Their assemblies were generated by conventional capillary The bioinformatics tools, together with the availability to sequencing resulting in 19.022 scaffolds (S. mansoni)and access parasite genome, should have helped the knowledge of 25.048 scaffolds (S. japonicum). More recently an improved parasite biology and the search for new vaccines, therapeutic version of the S. mansoni genome was published [5], utilizing targets, and antigens to be used in the disease diagnosis. In 2 Journal of Parasitology Research this paper we will present the advances in the development functions that ensure parasite survival [32], the study of its of vaccines and diagnostics tests achieved with the use of the structure and how it interacts with the host immune system information from schistosome genome, focus will be given can provide important information about disease control, to the parasite tegument as a source for antigens. especially to those related to the search for new drugs and vaccine development. We have recently demonstrated that 2. Host-Parasite Relationship: Role for the schistosomula tegument from S. mansoni (Smteg) is rec- the Parasite Tegument ognized by TLR4 in dendritic cells (DC) leading to DC activation and production of proinflammatory cytokines as IL- Highly adapted to parasitic life, schistosomes can live for 12 and TNF-α [33]. In contrast to this inflammatory profile, years or decades even in a hostile environment as the circu- Smteg also induce IL-10 production by DC in a TLR (Toll like latory system from vertebrate host where the parasite has an receptors) 2, 3, 4, and 9 independent manner (unpublished intimate contact with circulating elements of the immune data) once again demonstrating that schistosomula tegument system [8]. can both activate or modulate host immune system. In this successful host-parasite relationship, the host immune system plays an important role in both parasite 3. The Tegument as Antigen Source for development and elimination. CD4+ cells, hormones, and Vaccine Development cytokines as TNF-α,TGF-β, and IL-7 produced by the host, seem to assist the parasite development [9–15]. While CD4+ Most of the studies that aimed to identify membrane proteins cells, B cells, IFN-γ,andTNF-α has been described to be in parasite tegument were performed in adult worms [34– involved in parasite elimination in the irradiated cercariae 36]. Although schistosomula is the major target for host vaccine model [16–18]. immunity, its tegument proteins have still not been charac- Moreover, the highly adapted relationship between schis- terized, mainly due to the difficulty in obtaining sufficient tosomes and the mammalian definitive host also involves quantities of material for such protein studies [37]. Indeed the effective mechanisms for evading the immune response protective antigens are found in S. mansoni schistosomula that they provoke. In this context, the parasite tegument tegument (Smteg) since mice immunization with Smteg plays an important role [19, 20]. After penetration, the para- formulated with Freunds’ adjuvant [38]orAlum+CPG- site surface undergoes a profound change that allows parasite ODN (unpublished data) is able to reduce significantly worm adaptation into the host internal microenvironment where burden and egg elimination with the feces. The characteri- the parasite switches from its immune-sensitive to an zation of these protective antigens is being performed using immune-refractory state [21]. In cercariae, the surface is immune-proteomics analysis and genome databases to iden- characterized by a single bilayer membrane covered by a tify candidates to be used in a vaccine formulation against dense glicocalyx. During penetration, the glicocalyx is lost schistosomiasis. Other “omics” technologies are also being and the membrane transforms into a double bilayer mem- used to identify schistosoma proteins, mainly those expressed brane [22]. Evading mechanisms as antigenic mimicry, in schistosomula. In this context, two studies, using cDNA membrane turnover, production of immunomodulatory microarrays technologies assessed the most relevant tran- molecules and modulation of surface antigens expression scriptional changes in the schistosomula development phase. also takes place in the parasite surface and contributes to These studies demonstrated that tetraspanin, Sm22.6, Sm29, schistosome survival [23, 24]. Sm200 and phosphadiesterase are membrane proteins are Trying to eliminate the parasite, host immune system highly expressed during schistosomula phase [39, 40]. Fur- targets the antigens in parasite surface. Studies in mice have thermore, the studies that used gene silencing through RNAi shown that the developmental stage most susceptible to the technique could clarify the importance of some proteins, host immune system attack is the schistosomula stage. Very such as cathepsins [41, 42] and tetraspanins [43]forparasite early after infection, schistosomula are susceptible to cellular development and survival. The same membrane protein was and humoral immunity, however, in the course of parasite identified in adult worm tegument preparations using Mass development the susceptibility is rapidly lost [25, 26]. The spectrometry (MS-)-based proteomics [33, 34] together with resistance to host immune response acquired by parasites genome, transcriptome and genetic maps information [3, can be in part explained by surface changes independently 44–46]. Recently a proteomic analysis demonstrated that of host antigens adsorption [27–29]. In addition, El Ridi Sm29 and Sm200 are linked to parasite surface membrane and colleagues [30], demonstrated that lung-stage schisto- through a GPI-anchor [47] while the most abundant protein somulum protect themselves from the host immune system in adult worm tegument, among the investigated molecules, by confining antigenic molecules in lipid-rich sites of surface are aquaporin, dysferlin, TSP-2, and ATP diphosphohy- membrane. In contrast, McLaren, in 1989 [31], demon- drolase [48]. Among this expressive catalogue of protein strated that both skin and lung schistosomula phases are tar- expressed in the schistosome tegument, some of them have gets of the immune system in the radiation-attenuated vac- been evaluated as vaccine antigen in immunization protocols cine model which trigger an inflammatory reaction around in mice. The Table 1 summarizes the results observed in these the larvae inhibiting their migration. preclinical trials using tegument proteins. Since schistosomula is the major target of the host Sm29 was identified by Cardoso and coworkers using in immune system attack and its tegument represents the silico analysis to identify in S. mansoni transcriptome putative interface between parasite and host, also performing vital expressed proteins localized in the parasite tegument [49]. Journal of Parasitology Research 3

Table 1: Schistosome tegument protein evaluated as vaccine candidates in preclinical studies.

Bioinformatictoolusedin Protein Vaccine type Protection level Egg reduction References antigen selection Sm 21.7 Recombinant protein 41%–70% ND ND [63] 62% (liver) Sm 21.7 DNA vaccine 41.5% ND [64] 67% (intestine) Cu/Zn superoxide DNA vaccine 44%–60% ND ND [65] dismutase 64% (liver) Sm TSP2 Recombinant protein 57% BLAST [57, 83] 65% (feces) InterProScan, SignalIP 3.0, Signal IP Neural, NetNGlyc Sm29 Recombinant protein 51% 60% (intestine) [49, 50] 1.0, BLAST, WolfpSORT, SOSUI, Compute pI/Mw tool, ECL (200 kDa protein) DNA vaccine 38.1% ND ND [61] Sm 22.6 Recombinant protein 34.5% ND BLAST [53] 52% (liver) Sm TSP 1 Recombinant protein 34% BLAST [57, 83] 69% (intestine) ND: not determined.

Sm29 recombinant form induces a Th1 profile in mice asso- developmentally regulated. The lowest level of gene expres- ciated with a reduction of 51% in worm burden when used sion and enzyme-specific activity was found in the larval in vaccine formulation [50]. The tegumental protein, Sm22.6 stages while the highest level of gene expression was observed and its homologue in S. japonicum (Sj22.6), are involved in in adult worms [65–68]. This suggests that antioxidant resistance to reinfection in endemic areas [51, 52]. Immu- enzymes are important in immune evasion by adult schis- nization of mice with recombinant 22.6 formulated with Fre- tosome parasites [67]. Also RNAi assays demonstrated that und adjuvant resulted in 34.5% reduction on worm burden knocking down the antioxidants enzymes GPX and GST [53] while Sm22.6 formulated with alum failed to induce result in dramatic decreases in sporocysts survival indicating protection against schistosomiasis but induced a regulatory that these enzymes are capable of enhancing parasite survival response able to modulate allergic asthma in mice [54, 55]. in an oxidative environment [69]. Mice immunized with Tetraspanins (TSP) 1 and 2 were identified in a cDNA the antioxidant enzyme Cu-Zn superoxide dismutase in a library from S. mansoni based on their membrane-targeting DNA vaccine strategy resulted in 44–60% reduction in worm signal [56]. Immunization of mice with TSP1 recombinant burden [65]. protein resulted in a reduction of 57% in worm burden and reduction in the number of eggs in liver (64%) and 4. Antigens to Be Used in Schistosomiasis intestine (65%), TSP2 recombinant protein was less effective Diagnostic Test in reducing worm burden (34%) but had similar effects in reducing the number of eggs trapped in the liver (52%) and Currently, all available techniques for the diagnosis of schist- intestine (69%) [57]. The TSP-2 homologue in S. japonicum osomiasis are characterized by having some limitations, has also been evaluated in murine immunization however no especially when it becomes necessary to detect infection in protection was observed [58]. a large number of patients with low parasite load [70]. One ECL or Sm200 is a GPI-anchored protein in the S. man- of the initial difficulties in the development of a test for the soni tegument that has also been associated with praziquantel diagnosis of schistosomiasis is the choice of an appropriate efficacy, since antibodies against this protein can restore antigen. There are several factors that influence this choice: drug efficacy in B cells depleted mice [59, 60]. Murine DNA easily of production, high stability in sample storage, immu- vaccination with the gene encoding Sm200 elicited 38.1% nogenicity, specificity, and ability to be incorporated to low protection while immunization of mice with enzymatically costs test platforms [71]. cleaved GPI-anchored proteins from the S. mansoni tegu- In this context, the availability of the complete genome ment, in which Sm200 represent the most abundant protein sequences in combination with other technologies such as result in 43% reduction in adult worm burden [61, 62]. bioinformatics and proteomics, provides important tolls to Sm21.7 was tested as antigen in a recombinant vaccine [63] seek for an ideal candidate to compose an efficient immun- and DNA vaccine [64]. Immunization of mice with recom- odiagnostic test. With this in mind, our group have recently binant Sm21.7 resulted in a decrease of 41%–70% in worm designed an in silico strategy based in the principles of reverse burden while DNA vaccination resulted in of 41.5% worm vaccinology, and using a rational criteria to mine candidates burden reduction [63, 64]. in parasite genome to be used in the immunodiagnosis of The schistosome antioxidant enzymes (Cu/Zn super- schistosomiasis [72]. Six antigens were selected based on the oxide dismutase-SOD, glutathione-S-peroxidase-GPX) are evidence of gene expression at different phases of the parasite 4 Journal of Parasitology Research

Table 2: Schistosoma mansoni protein selected by genome mining to be used in serological diagnosis for schistosomiasis.

SchistoDB Number of Predicted Predicted Protein Annotation Base pairs Predicted location number amino acid molecular weight isoelectric point 200-kDa GPI-anchored Tegument surface Sm200 Smp 017730 1656 4971 186,5 kDa 4.97 surface glycoprotein membranes Sm12.8 Smp 034420.1 Expressed protein 117 354 12,8 kDa 6.88 Extracellular Sm43.5 Smp 042910 Expressed protein 382 1149 43,5 kDa 8.43 Extracellular Sm127.9 Smp 171300 Hypothetical protein 1143 3432 127,9 kDa 6.63 Extracellular Cytochrome oxidase Sm18.9 Smp 184440 171 516 18,9 kDa 9.30 Extracellular subunit, putative Cytochrome oxidase Sm16.5 Smp 184550 146 441 16,5 kDa 9.14 Extracellular subunit, putative Adapted of Carvalho et al., 2011 [72]. life cycle in the definitive host, accessibility to host immune organisms [76]. This protein was described to be abundant in system (exposed proteins), low similarity with human and schistosome tegument and due to its physiological function other helminthic proteins, and presence of predicted B cells and abundance represent an interesting target to vaccines epitopes (Table 2)[72]. Although our in silico analysis led to and diagnosis tests [48]. Characterization of the S. japonicum identification of six candidates, this strategy has not been yet aquaporin-3 using bioinformatics tools demonstrated that experimentally validated. this 32.9 kDa transmembrane protein has predicted B cells Other groups have also used bioinformatics analysis to epitopes with the most likely epitopes present in the N- select target sequence from S. japonicum genome to be used terminal portion of the protein, located outside the mem- for the detection of parasite DNA in blood samples. A 230- brane [77]. Other abundant protein in schistosoma tegument bp sequence from the highly repetitive retrotransposon SjR2 is dysferlin, based on analogy with homologues from other was identified and it was demonstrated that PCR test to organisms, this protein seems to be involved in membrane detect SjR2 is highly sensitive and specific for detection repair and/or vesicle fusion in tegument surface [34]. S. japonicum infection in the sera of infected rabbits and ATP-diphosphohydrolases are enzymes involved in ADP patients [73]. More recently the same group performed a and ATP hydrolysis that has been related to host immune sys- comparative study to determine the best target to be used tem evasion, since this enzyme could hydrolyze the ATP proin a molecular diagnosis test for schistosomiasis japonicum duced in response to parasite induced stress in the endothe- in 29 retrotransposons identified by bioinformatics analysis. lio thus modulating the DAMP (danger associated mole- A 303-bp sequence had the highest sensitivity and specificity cular pattern)-mediated inflammatory signaling [78, 79]. for the detection of S. japonicum DNA in serum samples [74]. In schistosomes two different proteins have been described Proteomics analysis has also been used in the identifica- SmATPDase 1 and SmATPDase2 with approximately 63 and tion of candidates to the immunodiagnosis of schistosomia- 55 kDa [80, 81]. SmATPDase 1 is located in the border sis. Western Blot with sera from S. japonicum infected rabbit of the tegument while SmATPDase2 is located in internal in a two-dimensional gel loaded with adult worm prepara- structure of the tegument syncytium and can be secreted tion identified 10 spots that were demonstrated by LC/MS- [81]. The immunogenicity of the synthetic peptide (r175– MS to correspond to four different proteins: SjLAP (Leucine 190) from SmATPDase2 has been demonstrated in Balb-c aminopeptidases), SjFBPA (fructose-1,6-bisphosphate aldo- mice, however the protection induced by this epitope has not lase), SjGST (Glutathione-S-transferase) and SJ22.6 [75]. been evaluated [82]. Recombinant SjLAP and SjFBPA were tested in ELISA assay Although most tegument protein listed in this paper has and presented high efficacy for the diagnosis of S. japonicum been identified in adult worm tegument, an in silico anal- infection, with 96.7% specificity for both proteins and 98.1% ysis performed in SchistoDB (http://www.schistodb.net/) or 87.8% sensitivity to detect acute and chronically infected demonstrates that some of them are also expressed in the individuals, respectively, when SjLAP was used as antigen or schistosomula stage as demonstrated in Figure 1 reinforcing a sensitivity of 100% (acute) and 84.7% (chronic infection) their potential to be used in a vaccine formulation or in the whenSjFBPAwasusedasantigen[75]. early diagnosis of schistosome infection.

6. Conclusion 5. Other Membrane Proteins Candidates to Be Used in Vaccine Formulation and So far the genome, transcriptome, and proteome informa- Diagnosis Tests tion provided many targets to be tested in schistosomiasis vaccine and diagnosis and also new knowledge about schisto- Aquaporins are small integral membrane proteins involved some biology. However approximately 40% of the schisto- in the selective transportation of water and other solutes some genome is composed of hypothetical proteins with through plasma membranes of mammals, plants and lower unknown function that represents interesting targets to be Journal of Parasitology Research 5

40 [8]A.R.C.Harris,R.J.Russell,andA.D.Charters,“Areviewof 35 schistosomiasis in immigrants in Western Australia, demonstrating the unusual longevity of Schistosoma mansoni,” Trans- 30 actions of the Royal Society of Tropical Medicine and Hygiene, 25 vol. 78, no. 3, pp. 385–388, 1984. 20 [9]S.J.Davies,J.L.Grogan,R.B.Blank,K.C.Lim,R.M.Lock- sley, and J. H. McKerrow, “Modulation of blood fluke develop- 15 ment in the liver by hepatic CD4+ lymphocytes,” Science, vol. Numbers ofNumbers EST 10 294, no. 5545, pp. 1358–1361, 2001. 5 [10] R. L. De Mendonça, H. Escriva,´ D. Bouton, V. Laudet, and R. J. Pierce, “Hormones and nuclear receptors in schistosome 0 development,” Parasitology Today, vol. 16, no. 6, pp. 233–240, Sm200 Sm29 TSP-2 TSP-1 Dysferlin Sm22.6 Sm21.7 2000. [11] P. Saule, E. Adriaenssens, M. Delacre et al., “Early variations Cercariae Adult worm of host thyroxine and interleukin-7 favor Schistosoma mansoni Schistosomula Egg development,” Journal of Parasitology, vol. 88, no. 5, pp. 849– 855, 2002. Figure 1: Predicted expression of schistosome tegument proteins [12] P.Amiri, R. M. Locksley, T. G. Parslow et al., “Tumour necrosis in the different parasite life stage in the definitive host. schistosome factor α restores granulomas and induces parasite egg-laying tegument protein identified by proteomics analysis of the adult in schistosome-infected SCID mice,” Nature, vol. 356, no. worm tegument was analyzed in SchistoDB database (http://www 6370, pp. 604–607, 1992. .schistodb.net/). Bars represent the numbers of EST in each parasite [13] P. T. LoVerde, A. Osman, and A. Hinck, “Schistosoma mansoni: life stage whose annotation correspond to Sm200, Sm29, TSP-2, TGF-β signaling pathways,” Experimental Parasitology, vol. TSP-1, Dysferlin, Sm22.6, or Sm21.7. 117, no. 3, pp. 304–317, 2007. [14] I. Wolowczuk, S. Nutten, O. Roye et al., “Infection of mice lacking interleukin-7 (IL-7) reveals an unexpected role for IL- tested and characterized. An increase in the knowledge about 7 in the development of the parasite Schistosoma mansoni,” parasite biology, pathogenesis, and host-parasite relationship Infection and Immunity, vol. 67, no. 8, pp. 4183–4190, 1999. can be expected for the next years. [15] R. B. Blank, E. W. Lamb, A. S. Tocheva et al., “The common γ chain cytokines interleukin (IL)-2 and IL-7 indirectly modulate blood fluke development via effects on CD4+ Tcells,” Acknowledgments Journal of Infectious Diseases, vol. 194, no. 11, pp. 1609–1616, 2006. This work was supported by CNPq, INCT-DT/CNPq, Ripag/ [16] D. A. A. Vignali, P. Crocker, Q. D. Bickle, S. Cobbold, H. Wald- CPqRR-Fiocruz, and Papes/Fiocruz. G. B. F. Carvalho and mann, and M. G. Taylor, “A role for CD4+ but not CD8+ T C. C. Alves both received fellowship from Fapemig. C. T. cells in immunity to Schistosoma mansoni induced by 20 krad- Fonseca received a fellowship from PQ/CNPq. irradiated and Ro 11-3128-terminated infections,” Immu- nology, vol. 67, no. 4, pp. 466–472, 1989. [17] D. Jankovic, T. A. Wynn, M. C. Kullberg et al., “Optimal vac- References cination against Schistosoma mansoni requires the induction of both B cell- and IFN-γ-dependent effector mechanisms,” [1] N. R. Bergquist, L. R. Leonardo, and G. F. Mitchell, “Vaccine- Journal of Immunology, vol. 162, no. 1, pp. 345–351, 1999. linked chemotherapy: can schistosomiasis control benefit [18] M. Street, P. S. Coulson, C. Sadler et al., “TNF is essential for from an integrated approach?” Trends in Parasitology, vol. 21, the cell-mediated protective immunity induced by the radi- no. 3, pp. 112–117, 2005. [2] N. Berhe, G. Medhin, B. Erko et al., “Variations in helminth ation-attenuated schistosome vaccine,” Journal of Immunol- faecal egg counts in Kato-Katz thick smears and their implica- ogy, vol. 163, no. 8, pp. 4489–4494, 1999. tions in assessing infection status with Schistosoma mansoni,” [19] F. G. C. Abath and R. C. Werkhauser, “The tegument of Acta Tropica, vol. 92, no. 3, pp. 205–212, 2004. Schistosoma mansoni: functional and immunological features,” [3] M. Berriman, B. J. Haas, P. T. Loverde et al., “The genome Parasite Immunology, vol. 18, no. 1, pp. 15–20, 1996. of the blood fluke Schistosoma mansoni,” Nature, vol. 460, no. [20] Z. G. Han, P. J. Brindley, S. Y. Wang, and C. Zhu, “Schistosoma 7253, pp. 352–358, 2009. genomics: new perspectives on schistosome biology and host- [4] Y. Zhou, H. Zheng, Y. Chen et al., “The Schistosoma japonicum parasite interaction,” Annual Review of Genomics and Human genome reveals features of host-parasite interplay,” Nature, Genetics, vol. 10, pp. 211–240, 2009. vol. 460, no. 7253, pp. 345–351, 2009. [21] M. K. Jones, G. N. Gobert, L. Zhang, P. Sunderland, and D. [5] A. V. Protasio, I. J. Tsai, A. Babbage et al., “A systematically P. McManus, “The cytoskeleton and motor proteins of human improved high quality genome and transcriptome of the schistosomes and their roles in surface maintenance and host- human blood fluke Schistosoma mansoni,” PLoS Neglected parasite interactions,” BioEssays, vol. 26, no. 7, pp. 752–765, Tropical Diseases, vol. 6, no. 1, Article ID e1455, 2012. 2004. [6] N. D. Young, A. R. Jex, B. Li et al., “Whole-genome sequence [22] D. J. Hockley and D. J. McLaren, “Schistosoma mansoni: of Schistosoma haematobium,” Nature Genetics,vol.44,no.2, changes in the outer membrane of the tegument during pp. 221–225, 2012. development from cercaria to adult worm,” International Jour- [7] A. Zerlotini, M. Heiges, H. Wang et al., “SchistoDB: a Schisto- nal for Parasitology, vol. 3, no. 1, pp. 13–20, 1973. soma mansoni genome resource,” Nucleic Acids Research, vol. [23] M. Salzet, A. Capron, and G. B. Stefano, “Molecular crosstalk 37, no. 1, pp. D579–D582, 2009. in host-parasite relationships: Schistosome- and leech-host 6 Journal of Parasitology Research

interactions,” Parasitology Today, vol. 16, no. 12, pp. 536–540, [40] G. N. Gobert, M. H. Tran, L. Moertel et al., “Transcriptional 2000. changes in Schistosoma mansoni during early schistosomula [24] R. T. Damian, “Molecular mimicry revisited,” Parasitology development and in the presence of erythrocytes,” PLoS Today, vol. 3, no. 9, pp. 263–266, 1987. Neglected Tropical Diseases, vol. 4, no. 2, article e600, 2010. [25] S. R. Smithers, D. J. McLaren, and F. J. Rahalho-Pinto, “Immu- [41]J.M.Correnti,P.J.Brindley,andE.J.Pearce,“Long-termsup- nity to schistosomes: the target,” American Journal of Tropical pression of cathepsin B levels by RNA interference retards Medicine and Hygiene, vol. 26, no. 6, pp. 11–19, 1977. schistosome growth,” Molecular and Biochemical Parasitology, [26] F. Santoro, P. J. Lachmann, A. Capron, and M. Capron, “Acti- vol. 143, no. 2, pp. 209–215, 2005. vation of complement by Schistosoma mansoni schistosomula: [42] M. E. Morales, G. Rinaldi, G. N. Gobert, K. J. Kines, J. F. Tort, killing of parasites by the alternative pathway and requirement and P. J. Brindley, “RNA interference of Schistosoma mansoni of IgG for classical pathway activation,” Journal of Immunol- cathepsin D, the apical enzyme of the hemoglobin proteolysis ogy, vol. 123, no. 4, pp. 1551–1557, 1979. cascade,” Molecular and Biochemical Parasitology, vol. 157, no. [27] D. A. Dean, “Decreased binding of cytotoxic antibody by 2, pp. 160–168, 2008. developing Schistosoma mansoni. Evidence for a surface [43]M.H.Tran,T.C.Freitas,L.Cooperetal.,“Suppressionof change independent of host antigen adsorption and mem- mRNAs encoding tegument tetraspanins from Schistosoma brane turnover,” Journal of Parasitology, vol. 63, no. 3, pp. 418– mansoni results in impaired tegument turnover,” PLoS path- 426, 1977. ogens, vol. 6, no. 4, Article ID e1000840, 2010. [28]A.Dessein,J.C.Samuelson,andA.E.Butterworth,“Immune [44] F. Liu, S. J. Cui, W. Hu, Z. Feng, Z. Q. Wang, and Z. G. Han, evasion by Schistosoma mansoni: loss of susceptibility to anti- “Excretory/secretory proteome of the adult developmental body or complement-dependent eosinophil attack by schisto- stage of human blood fluke, Schistosoma japonicum,” Molecu- somula cultured in medium free of macromolecules,” Para- lar and Cellular Proteomics, vol. 8, no. 6, pp. 1236–1251, 2009. sitology, vol. 82, no. 3, pp. 357–374, 1981. [45] S. Verjovski-Almeida, R. DeMarco, E. A. L. Martins et al., [29] E. L. Racoosin, S. J. Davies, and E. J. Pearce, “Caveolae-like “Transcriptome analysis of the acoelomate human parasite structures in the surface membrane of Schistosoma mansoni,” Schistosoma mansoni,” Nature Genetics, vol. 35, no. 2, pp. 148– Molecular and Biochemical Parasitology, vol. 104, no. 2, pp. 157, 2003. 285–297, 1999. [46] C. D. Criscione, C. L. L. Valentim, H. Hirai, P. T. LoVerde, [30] R. El Ridi, S. H. Mohamed, and H. Tallima, “Incubation and T. J. C. Anderson, “Genomic linkage map of the human of Schistosoma mansoni lung-stage schistosomula in corn oil blood fluke Schistosoma mansoni,” Genome Biology, vol. 10, no. exposes their surface membrane antigenic specificities,” Jour- 6, article R71, 2009. nal of Parasitology, vol. 89, no. 5, pp. 1064–1067, 2003. [47] W. Castro-Borges, A. Dowle, R. S. Curwen, J. Thomas-Oates, [31] D. J. McLaren, “Will the real target of immunity to schistoso- and R. A. Wilson, “Enzymatic shaving of the tegument sur- miasis please stand up,” Parasitology Today, vol. 5, no. 9, pp. face of live schistosomes for proteomic analysis: a rational 279–282, 1989. approach to select vaccine candidates,” PLoS Neglected Tropical [32] G. N. Gobert, M. Chai, and D. P. McManus, “Biology of Diseases, vol. 5, no. 3, article e993, 2011. the schistosome lung-stage schistosomulum,” Parasitology, vol. 134, no. 4, pp. 453–460, 2007. [48] W. Castro-Borges, D. M. Simpson, A. Dowle et al., “Abun- [33] F. V. Duraes,˜ N. B. Carvalho, T. T. Melo, S. C. Oliveira, and dance of tegument surface proteins in the human blood fluke C. T. Fonseca, “IL-12 and TNF-α production by dendritic cells Schistosoma mansoni determined by QconCAT proteomics,” stimulated with Schistosoma mansoni schistosomula tegument Journal of Proteomics, vol. 74, pp. 1519–1533, 2011. is TLR4- and MyD88-dependent,” Immunology Letters, vol. [49] F. C. Cardoso, J. M. R. Pinho, V. Azevedo, and S. C. Oliveira, 125, no. 1, pp. 72–77, 2009. “Identification of a new Schistosoma mansoni membrane- [34] B. W. M. Van Balkom, R. A. Van Gestel, J. F. H. M. Brouwers et bound protein through bioinformatic analysis,” Genetics and al., “Mass spectrometric analysis of the Schistosoma mansoni Molecular Research, vol. 5, no. 4, pp. 609–618, 2006. tegumental sub-proteome,” Journal of Proteome Research, vol. [50] F. C. Cardoso, G. C. Macedo, E. Gava et al., “Schistosoma man- 4, no. 3, pp. 958–966, 2005. soni tegument protein Sm29 is able to induce a Th1-type of [35] S. Braschi, W. C. Borges, and R. A. Wilson, “Proteomic analysis immune response and protection against parasite infection,” of the shistosome tegument and its surface membranes,” PLoS Neglected Tropical Diseases, vol. 2, no. 10, article e308, Memorias do Instituto Oswaldo Cruz, vol. 101, supplement 1, 2008. pp. 205–212, 2006. [51] D. W. Dunne, M. Webster, P. Smith et al., “The isolation of [36] S. Braschi and R. A. Wilson, “Proteins exposed at the adult a 22 kDa band after SDS-PAGE of Schistosoma mansoni adult schistosome surface revealed by biotinylation,” Molecular and worms and its use to demonstrate the IgE responses against Cellular Proteomics, vol. 5, no. 2, pp. 347–356, 2006. the antigen(s) it contains are associated with human resistance [37] A. Loukas, S. Gaze, J. P. Mulvenna et al., “Vaccinomics for the to reinfection,” Parasite Immunology, vol. 19, no. 2, pp. 79–89, major blood feeding helminths of humans,” OMICS A Journal 1997. of Integrative Biology, vol. 15, no. 9, pp. 567–577, 2011. [52] M. L. Santiago, J. C. R. Hafalla, J. D. Kurtis et al., “Identifica- [38] T. Teixeira De Melo, J. Michel De Araujo, F. Do Valle Duraes˜ tion of the Schistosoma japonicum 22.6-kDa antigen as a major et al., “Immunization with newly transformed Schistosoma target of the human IgE response: similarity of IgE-binding mansoni schistosomula tegument elicits tegument damage, epitopes to allergen peptides,” International Archives of Allergy reduction in egg and parasite burden,” Parasite Immunology, and Immunology, vol. 117, no. 2, pp. 94–104, 1998. vol. 32, no. 11-12, pp. 749–759, 2010. [53] L. G. G. Pac´ıfico, C. T. Fonseca, L. Chiari, and S. C. Oliveira, [39] G. P.Dillon, T. Feltwell, J. P.Skelton et al., “Microarray analysis “Immunization with Schistosoma mansoni 22.6 kDa antigen identifies genes preferentially expressed in the lung schisto- induces partial protection against experimental infection in a somulum of Schistosoma mansoni,” International Journal for recombinant protein form but not as DNA vaccine,” Immu- Parasitology, vol. 36, no. 1, pp. 1–8, 2006. nobiology, vol. 211, no. 1-2, pp. 97–104, 2006. Journal of Parasitology Research 7

[54] L. G. G. Pac´ıfico, C. T. Fonseca, M. M. Barsante, L. S. Cardoso, enzymes,” Experimental Parasitology, vol. 86, no. 1, pp. 69–78, M. I. Araujo,´ and S. C. Oliveira, “Aluminum hydroxide asso- 1997. ciated to Schistosoma mansoni 22.6 kDa protein abrogates [68]H.Mei,A.Thakur,J.Schwartz,andP.T.LoVerde,“Expression partial protection against experimental infection but not alter and characterization of glutathione peroxidase activity in interleukin-10 production,” Memorias do Instituto Oswaldo the human blood fluke Schistosoma mansoni,” Infection and Cruz, vol. 101, supplement 1, pp. 365–368, 2006. Immunity, vol. 64, no. 10, pp. 4299–4306, 1996. [55] L. S. Cardoso, S. C. Oliveira, A. M. Goes´ et al., “Schistosoma [69] M. D. M. Mourao,˜ N. Dinguirard, G. R. Franco, and T. P. mansoni antigens modulate the allergic response in a murine Yoshino, “Role of the endogenous antioxidant system in the model of ovalbumin-induced airway inflammation,” Clinical protection of Schistosoma mansoni primary sporocysts against and Experimental Immunology, vol. 160, no. 2, pp. 266–274, exogenous oxidative stress,” PLoS Neglected Tropical Diseases, 2010. vol. 3, no. 11, article e550, 2009. [56] D. Smyth, D. P. McManus, M. J. Smout, T. Laha, W. Zhang, [70] J. V. Hamilton, M. Klinkert, and M. J. Doenhoff, “Diagnosis of and A. Loukas, “Isolation of cDNAS encoding secreted and schistosomiasis: antibody detection, with notes on parasito- transmembrane proteins from Schistosoma mansoni by a signal logical and antigen detection methods,” Parasitology, vol. 117, sequence trap method,” Infection and Immunity,vol.71,no.5, pp. S41–S57, 1998. pp. 2548–2554, 2003. [71] M. J. Doenhoff, P. L. Chiodini, and J. V. Hamilton, “Specific [57] M. H. Tran, M. S. Pearson, J. M. Bethony et al., “Tetraspanins and sensitive diagnosis of schistosome infection: can it be done on the surface of Schistosoma mansoni are protective antigens with antibodies?” Trends in Parasitology, vol. 20, no. 1, pp. 35– against schistosomiasis,” Nature Medicine, vol. 12, no. 7, pp. 39, 2004. 835–840, 2006. [72]G.B.F.Carvalho,R.A.daSilva-Pereira,L.G.G.Pac´ıfico, and [58] W. Zhang, J. Li, M. Duke et al., “Inconsistent protective effi- C. T. Fonseca, “Identification of Schistosoma mansoni candi- cacy and marked polymorphism limits the value of Schisto- date antigens for diagnosis of schistosomiasis,” Memorias do soma japonicum tetraspanin-2 as a vaccine target,” PLoS Neg- Instituto Oswaldo Cruz, vol. 106, no. 7, pp. 837–843, 2011. lected Tropical Diseases, vol. 5, no. 5, Article ID e1166, 2011. [73] C. M. Xia, R. Rong, Z. X. Lu et al., “Schistosoma japonicum:a [59] S. Y. Sauma and M. Strand, “Identification and characteriza- PCR assay for the early detection and evaluation of treatment tion of glycosylphosphatidylinositol-linked Schistosoma man- in a rabbit model,” Experimental Parasitology, vol. 121, no. 2, soni adult worm immunogens,” Molecular and Biochemical pp. 175–179, 2009. Parasitology, vol. 38, no. 2, pp. 199–210, 1990. [74] J.-J. Guo, H.-J. Zheng, J. Xu, X.-Q. Zhu, S.-Y. Wang, and C.- [60] P. J. Brindley, M. Strand, A. P. Norden, and A. Sher, “Role of M. Xia, “Sensitive and specific target sequences selected from host antibody in the chemotherapeutic action of praziquantel retrotransposons of Schistosoma japonicum for the diagnosis against Schistosoma mansoni: identification of target antigens,” of schistosomiasis,” PLoS Neglected Tropical Diseases, vol. 6, no. Molecular and Biochemical Parasitology, vol. 34, no. 2, pp. 99– 3, Article ID e1579, 2012. 108, 1989. [75] Z. R. Zhong, H. B. Zhou, X. Y. Li et al., “Serological pro- [61] E. J. M. Nascimento, R. V. Amorim, A. Cavalcanti et al., teome-oriented screening and application of antigens for the “Assessment of a DNA vaccine encoding an anchored- gly- diagnosis of Schistosomiasis japonica,” Acta Tropica, vol. 116, cosylphosphatidylinositol tegumental antigen complexed to no. 1, pp. 1–8, 2010. protamine sulphate on immunoprotection against murine [76] A. S. Verkman, “Physiological importance of aquaporin water schistosomiasis,” Memorias do Instituto Oswaldo Cruz, vol. channels,” Annals of Medicine, vol. 34, no. 3, pp. 192–200, 102, no. 1, pp. 21–27, 2007. 2002. [62] V. P. Martins, C. S. Pinheiro, B. C. P. Figueiredo et al., “Vac- [77] J. Song and Q.-F. He, “Bioinformatics analysis of the structure cination with enzymatically cleaved GPI-anchored proteins and linear B-cell epitopes of aquaporin-3 from Schistosoma from schistosoma mansoni induces protection against chal- japonicum,” Asian Pacific Journal of Tropical Medicine, vol. 5, lenge infection,” Clinical and Developmental Immunology, vol. no. 2, pp. 107–109, 2012. 2012, Article ID 962538, 11 pages, 2012. [78] E. G. Vasconcelos, P. S. Nascimento, M. N. L. Meirelles, S. [63] H. M. Ahmed and M. H Romeih, “Protection against Schis- Verjovski-Almeida, and S. T. Ferreira, “Characterization and tosoma mansoni infection with recombinant schistosomula localization of an ATP-diphosphohydrolase on the external 21.7 kDa protein,” Arab Journal of Biotechnology, vol. 24, pp. surface of the tegument of Schistosoma mansoni,” Molecular 229–249, 2001. and Biochemical Parasitology, vol. 58, no. 2, pp. 205–214, 1993. [64] H. M. Ahmed, M. H. Romeih, and T. S. Abou-Shousha, “DNA [79] R. Bhardwaj and P.J. Skelly, “Purinergic signaling and immune immunization with the gene encoding Sm21.7 protects mice modulation at the schistosome surface?” Trends in Parasito- against S. mansoni infections,” American Journal of Science, vol. logy, vol. 25, no. 6, pp. 256–260, 2009. 2, pp. 59–69, 2006. [80] R. DeMarco, A. T. Kowaltowski, R. A. Mortara, and S. [65] K. A. Shalaby, L. Yin, A. Thakur, L. Christen, E. G. Niles, and P. Verjovski-Almeida, “Molecular characterization and immu- T. LoVerde, “Protection against Schistosoma mansoni utilizing nolocalization of Schistosoma mansoni ATP-diphosphohydro- DNA vaccination with genes encoding Cu/Zn cytosolic super- lase,” Biochemical and Biophysical Research Communications, oxide dismutase, signal peptide-containing superoxide dismu- vol. 307, no. 4, pp. 831–838, 2003. tase and glutathione peroxidase enzymes,” Vaccine, vol. 22, no. [81] J. Levano-Garcia, R. A. Mortara, S. Verjovski-Almeida, and 1, pp. 130–136, 2003. R. DeMarco, “Characterization of Schistosoma mansoni ATP- [66]Z.Hong,D.J.Kosman,A.Thakur,D.Rekosh,andP.T. Dase2 gene, a novel apyrase family member,” Biochemical and LoVerde, “Identification and purification of a second form Biophysical Research Communications, vol. 352, no. 2, pp. 384– of Cu/Zn superoxide dismutase from Schistosoma mansoni,” 389, 2007. Infection and Immunity, vol. 60, no. 9, pp. 3641–3651, 1992. [82]R.G.P.R.Mendes,M.A.N.Gusmao,A.C.R.G.Maia˜ [67] H. Mei and P. T. LoVerde, “Schistosoma mansoni:thedevel- et al., “Immunostimulatory property of a synthetic peptide opmental regulation and immunolocalization of antioxidant belonging to the soluble ATP diphosphohydrolase isoform 8 Journal of Parasitology Research

(SmATPDase 2) and immunolocalisation of this protein in the schistosoma mansoni egg,” Memorias do Instituto Oswaldo Cruz, vol. 106, no. 7, pp. 808–813, 2011. [83] M. E. Hemler, “Speciﬁc tetraspanin functions,” JournalofCell Biology, vol. 155, no. 7, pp. 1103–1107, 2001. Hindawi Publishing Corporation Journal of Parasitology Research Volume 2012, Article ID 761968, 9 pages doi:10.1155/2012/761968

Review Article Pathogenicity of Trichobilharzia spp. for Vertebrates

Lichtenbergova´ Lucie and Horak´ Petr

Department of Parasitology, Faculty of Science, Charles University in Prague, Viniˇcna´ 7, 128 44 Prague 2, Czech Republic

Correspondence should be addressed to Lichtenbergova´ Lucie, [email protected]

Received 13 July 2012; Accepted 13 September 2012

Academic Editor: Rashika El Ridi

Copyright © 2012 L. Lucie and H. Petr. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Bird schistosomes, besides being responsible for bird schistosomiasis, are known as causative agents of cercarial dermatitis. Cercarial dermatitis develops after repeated contact with cercariae, mainly of the genus Trichobilharzia, and was described as a type I, immediate hypersensitivity response, followed by a late phase reaction. The immune response is Th2 polarized. Primary infection leads to an inﬂammatory reaction that is insuﬃcient to eliminate the schistosomes and schistosomula may continue its migration through the body of avian as well as mammalian hosts. However, reinfections of experimental mice revealed an immune reaction leading to destruction of the majority of schistosomula in the skin. Infection with the nasal schistosome Trichobilharzia regenti probably represents a higher health risk than infections with visceral schistosomes. After the skin penetration by the cercariae, parasites migrate via the peripheral nerves, spinal cord to the brain, and terminate their life cycle in the nasal mucosa of waterfowl where they lay eggs. T. regenti can also get over skin barrier and migrate to CNS of experimental mice. During heavy infections, neuroinfections of both birds and mammals lead to the development of a cellular immune response and axonal damage in the vicinity of the schistosomulum. Such infections are manifest by neuromotor disorders.

1. Introduction to target organs [2, 3]. In mammals, bird schistosomes can survive for several days or weeks, but they never mature Despite their worldwide distribution, avian schistosomes [4, 5]. The exact reason why bird schistosomes die in mam- were neglected by parasitologists who assumed that they malian hosts has not been known until the present. have no or minor pathogenic impact on birds or mammals, Studies on bird schistosomes disclosed a new species— including humans. Nowadays, many studies focus on these Trichobilharzia regenti [4]— with unusual behavior in com- parasites since it has been recognized that they can be severe patible as well, noncompatible hosts. In comparison to the pathogens of birds. Moreover, their larval stages (cercariae) majority of bird schistosome species living in the blood frequently infect humans and cause cercarial dermatitis. The system of visceral organs, mature T. regenti occur in the most reported agents of swimmer’s itch are cercariae of the nasals of their definitive host where they lay eggs. Migration genus Trichobilharzia [1]. of the worms from the skin to the nasals is via the spinal cord Human infections by bird schistosomes are associated and brain [4]. Experimental infections of mice showed that T. mostly with the development of cercarial dermatitis (swim- regenti schistosomula can evade attack by immune cells in the mer’s itch), an allergic skin response, which develops after skin of mammalian hosts allowing them to migrate further repeated contact with cercariae penetrating into the skin. For through the central nervous system (CNS) where immature a long time, it was assumed that the reaction eliminated the worms die after several days [5, 6]. Migration of the parasites majority of the schistosomes that had penetrated into the through CNS of both bird and mammalian hosts causes skin. However, the studies on mice infected experimentally severe tissue injuries [6, 7] that can result in leg paralysis, with bird schistosomes showed that soon after the penetra- balance, and orientation disorders and even host death [4, 7]. tion, the cercariae transform to schistosomula. Under certain Nowadays, mainly two species of bird schistosomes, T. circumstances, these schistosomula are able to resist host szidati and T. regenti, are studied under laboratory conditions immune response, escape from the skin, and migrate further with regard to their development, physiology (including 2 Journal of Parasitology Research enzymes participating in host tissue degradation and diges- represent an adaptation to invade duck skin that has a lower tion), immunomodulation of the host immune response, content of free fatty acids compared to human skin [20]. and pathogenicity towards natural and accidental hosts. Therefore, human skin with higher amount of surface lipids In field studies, the emphasis is on the study of species is likely more attractive to bird schistosome cercariae than spectrum, inter- and intraspecific variability, and prevalence duck skin [19]. of bird schistosomes (see, e.g., Brant and Loker [8], Jouet Penetration through the skin is facilitated by a number et al. [9], and Korsunenko et al. [10]; see also review by ofproteolyticenzymes,whicharereleasedfromcercarial Horak´ and Kola´rovˇ a[´ 1]). With regard to T. szidati and circum- and postacetabular glands immediately after attach- T. regenti, their occurrence has been reported from several ing to the host skin. In the case of bird schistosomes, countries. In particular, T. regenti cercariae have been found glandular secretion is stimulated mainly by fatty acids, in freshwater ponds for example, in Russia [11] and cercariae ceramides, and cholesterol [20]. Cercarial glands fill about of T. szidati in Russia, Belorussia [11], Germany [12]and one-third of the cercarial body [21] and contain many France [13]. Several findings of T. szidati infections in birds of the potentially antigenic proteins. The most important were reported, for example, from France [9], Poland, and penetration enzyme of S. mansoni is probably a serine Czech Republic [14]. Infections of birds with T. regenti were protease, elastase [22]. Nevertheless, Mikesˇ et al. [23]and detected for example, in Iceland [15] and in France, where Kasnˇ y´ et al. [24] did not find any elastase activity in the the prevalence on three studied localities reached 40% [9]. secretions of T. szidati and T. regenti cercariae, and it was Based on findings of Rudolfova´ et al. [14, 16], prevalence of not found in the congener S. japonicum [25]. However, T. regenti infection of waterfowl was 14% in Czech Republic cathepsin B-like activity was detected in the aforementioned (one studied locality) [16] and 22% in Gdansk area in Poland species. This enzyme from cercarial penetration glands is [14]. Although T. szidati cercariae are mostly distributed considered to be the main component in the cercarial throughout Europe, there is a report of their occurrence in penetration process [23–25].Thesametypesofenzymes snails collected from Michigan and Montana in the United could be the reason for similar penetration speed of S. States [8]. japonicum and Trichobilharzia cercariae [20]. In addition, six The main aim of our review is to summarize the present isoforms of cathepsin B1 (TrCB1.1–TrCB1.6) and cathepsin knowledge of the pathogenesis of bird schistosomiasis and B2 (TrCB2) were identified in an extract of migrating T. the immune reactions to bird schistosomes presence in avian regenti schistosomula [26, 27]. Two isoforms, TrCB1.1 and and mammalian hosts, with a special emphasis on T. regenti. TrCB1.4, degrade myelin basic protein, but do not efficiently The neurotropic species T. regenti,duetoitsunusualmodeof cleave hemoglobin [26]. The recombinant form of TrCB2 migration and potential pathogenic impact on avian as well is able to cleave protein components of the skin (keratin, as mammalian hosts, deserves more attention. Therefore, collagen, and elastin) as well as nervous tissue (myelin basic a major part of this review is dedicated to this species of protein), but has negligible activity towards hemoglobin schistosome. [27].Theenzymecould,therefore,serveasatoolfor migration through the host skin and subsequently through 2. Skin Infection the nervous tissue. Host fatty acids seem to stimulate not only the penetra- After leaving the snail intermediate hosts, bird schistosome tion of cercaria through the host skin, but also transforma- cercariae have a tendency to cling to the water surface and tion of their tegument as a part of parasite immune evasion wait for their definitive host. They react to shadow stimuli [19]. Penetration of the cercariae into the host skin is and start to swim with a negative phototactic orientation accompanied by cercaria/schistosomulum transformation from the water surface toward the definitive host [17]. Except with reconstruction of tegumental surface. Transformation for physical stimuli such as shadow, water turbulence, and starts with loss of tail, a process supported by a sphincter warmth, cercariae respond to host chemical cues like duck- muscle in cercarial hindbody [19], then the cercariae shed foot skin lipids—cholesterol and ceramides [17, 18]. the glycocalyx and start to form a surface double membrane. Once attached to the host skin, cercariae creep on the skin Creation of a new surface is accompanied by the disappear- and search for a suitable penetration site [19]. In contrast ance of lectin and antibody targets on the surface of the to human schistosomes, such as Schistosoma mansoni,which schistosomula [28]. penetrate smooth skin, bird schistosome cercariae prefer In the skin of the bird hosts, schistosomula move through skin wrinkles and hair follicles for penetration [20]. Studies the skin towards deeper layers and, therefore, require infor- on cercarial behavior of S. mansoni and T. szidati revealed mation for orientation. Studies on visceral schistosomes differences in the speed of migration through the host skin. invading humans, S. mansoni,andbirds,T. ocellata, showed For example, cercariae of T. szidati invade human skin more that schistosomula use negative photo orientation to move efficiently than S. mansoni such that they are able to locate away from light source [29]. The other stimulus involved entry sites and penetrate through the skin more rapidly than in navigation of the visceral schistosomula is represented by S. mansoni [20]. the concentration gradient of chemicals, such as D-glucose Skin penetration by cercariae is stimulated by fatty acids and L-arginine [30]. Unfortunately, data about orientation [19]. According to the study of Haas and Haeberlein [20], of the nasal species T. regenti are not complete, but there is an T. szidati cercariae respond to linolenic acid with higher indication that the stimuli differ from those used by visceral sensitivity if compared to S. mansoni. This feature seems to species (unpublished). Journal of Parasitology Research 3

3. Cercarial Dermatitis accompanied by a transient release of acute phase cytokines (IL-1β and IL-6) and increasing amounts of IL-12 [2]. In humans, the skin infection is a result of the development Increased production of IL-12 in the skin correlated with of an inflammatory reaction known as swimmer’s itch or higher Th1-associated IFN-γ production by cells from skin- cercarial dermatitis. Cercarial dermatitis can occur after draining lymph nodes [2]. Similarly, the study of Hogg et al. contact with water containing cercariae from snails infected [40] using S. mansoni illustrates rapid host immune response by bird schistosomes. Chances of getting cercarial dermatitis to parasite penetration with production of acute phase increase with repeated exposures to the parasite. Higher cytokines, such as IL-1β and IL-6 which were detected in the incidence of the infection is connected with bathing in supernatants of skin biopsies from wild-type mice. Both IL- shallow water, which is the preferred habitat for water snails 1β and IL-6 promote Th17-cell differentiation [41], therefore and, therefore, a place where cercariae accumulate [31]. implying that primary mouse infection with human as well Penetration of cercariae into the skin may result in an as avian schistosomes induces a Th17 polarized response. immediate prickling sensation that lasts for about 1 hour Skin immune response to challenge infections leads [32]. Severity and intensity of cercarial dermatitis depend to capture and elimination of the majority of schistoso- on various factors including the number and duration of mula in the skin [2]. In the early phase after re-infec- exposures to the cercariae, and host immune status, that is, tion with T. regenti cercariae, infiltration of mouse skin history of cercarial dermatitis, and individual susceptibility with inflammatory cells (high density of granulocytes and to the infection [32]. After a primary infection, the skin neutrophils, abundant MHC II APCs, macrophages and reaction is unapparent or mild with small and transient CD4+ lymphocytes) was also accompanied by oedema caus- macules or maculopapules, which develop after 5–14 days ed by local vascular permeability that was initiated by [32]. The most pronounced disease occurs after repeated histamine produced by activated mast cells and basophils exposures that result in a strong inflammatory reaction [2]. Degranulation of mast cells and basophils with release against the parasites [33]. The skin disease manifests by of histamine and IL-4 is realized after binding of IgE-antigen maculo-papulovesicular eruptions accompanied by intense complex via high affinity receptors FcεRI on the cell surface itching and, occasionally, by erythema, fever, local lymph [42, 43]. Histamine has been described previously as a potent node swelling, oedema. Massive infections may also cause effector of Th1 and Th2 responses as well as immunoglobulin nausea and diarrhea (for a review see Horak´ et al. [34]). Skin synthesis [44, 45]. Repeated infections with T. regenti evoke lesions develop only on those parts of body where there was dominant production of Th2-type cytokines, and the first cercarial penetration [35]. and most abundant cytokine detected in supernatants of skin Diagnosis of cercarial dermatitis is based on anamnesis biopsies from mice after repeated infections is IL-6 [2], which and clinical findings [36]. Some work has been done using can initiate Th2-type polarization via induction of IL-4 [46]. serological tests for confirmation of the diagnosis [37]. Within 1 hour after the penetration by T. regenti cercariae a Nevertheless, immunological tests are not routinely available massive upregulation of IL-4 and IL-10 can be observed in and laboratory confirmation of causative agents of the the skin biopsies, and the level of these cytokines declines dermatitis remains difficult. after 48 h. This upregulation during T. regenti infection is accompanied by release of histamine and proliferation of 4. Skin Immune Response mast cells [2]. Production of histamine and IL-4 detected in skin biopsies immediately after the last infection of re- Clinical pattern of cercarial dermatitis is linked with histo- infected mice was realized via IgE-dependent mast cell pathological reactions to the infection. In the case of the degranulation [2]. IL-4 plays also a crucial role in the human schistosome, Schistosoma mansoni, infections in development of Th2-type immune responses to S. mansoni naive mice led to a mild skin response with contribution antigens and regulation of immunoglobulin isotype switch to of neutrophils and mononuclear cells. In contrast, a more IgE [47]. CD4+ cells, numbers of which significantly increase severe cellular reaction developed in the mouse skin after in the skin after challenge infections, are potential sources of repeated infections with S. mansoni [38]. Similarly as for IL-4 associated with Th2 response [2]. human schistosomes, infections of mice with the bird Like mast cells, basophils possess high-affinity IgE sur- schistosomes T. szidati (T. ocellata)andT. regenti initiate the face receptors (FcεRI) that, after antigen-specific cross- development of a skin immune response [2, 39]. Primary link, induce production and release of mediators such as mouse infection with T. regenti initiates an acute inflamma- histamine and IL-4 [48]. In vitro stimulation of basophils tion with oedema, vasodilatation, and tissue infiltration by obtained from healthy (nonsensitized) humans by homo- neutrophils, macrophages, mast cells, and MHC II antigen genate of cercariae and excretory/secretory (E/S) products presenting cells (APCs), and a weak infiltration by CD4+ of T. regenti cercariae revealed that these antigens induce lymphocytes; repeated infections cause substantially elevated basophil degranulation and release of IL-4 [49]. Antigens infiltration of all cells mentioned above [2, 6]. Trichobilharzia stimulated basophil release of IL-4 in a dose-dependent regenti primoinfection leads to the development of an manner, and antigens from E/S products were more potent inflammatory reaction in the murine skin within 1–6 h after inducers of IL-4 release than cercarial homogenate [49]. exposure [2]. Detection of cytokine production by in vitro Elevated levels of skin mast cells [2] and high titers of serum cultured biopsies of pinnae (later skin biopsies) obtained IgE in mice infected repeatedly with T. regenti [49] showed from primoinfected mice revealed that the inflammation is that the cells of mast cell/basophil lineage play an important 4 Journal of Parasitology Research role in development of Th2 responses during Trichobilharzia recorded [50]. In schistosomula, spherical bodies represented infections. the most reactive structure. Further development of the parasite was accompanied with loss of immunoreactivity. In adult worms, the antibodies recognized the surface and sub- 5. Antibody Response and Antigens of tegumental cells, but with lower intensity if compared to the Bird Schistosomes larval stages [50]. Spherical bodies primary located within subtegumental Domination of Th-2 polarization of the immune response cells were transferred via cytoplasmic bridges to the surface after repeated T. regenti infections was confirmed by meas- of schistosomula. As in human schistosomes [56], these urement of antigen-specific antibody levels. Similarly as in bodies probably release their content on surface of the the study of Kourilovˇ a´ et al. [2], the increase of Th2-asso- tegument where the antigenic molecules could be recognized ciated antigen-specific IgG1 and total serum IgE antibodies by the host immune system [50]. However, composition of with concurrent decline of Th1-associated IgG2b antibody the spherical body content is not known till present. was demonstrated in sera of mice repeatedly infected with T. Nevertheless, based on Western blot analysis of cercarial regenti [49]. homogenate probed with sera from re-infected mice, several During T. regenti primary infection, IgM response antigens (14.7, 17, 28, 34, and 50 kDa) were identified by against glycan structures of the cercariae and their excre- IgG and IgE antibodies. Antigens of 34 and 50 kDa were tory/secretory (E/S) products was observed [49]. This also recognized in cercarial gland secretions [49]. A precise indicates that early antibody response is directed against identification of the 34 kDa molecule which is regarded as a components of highly antigenic cercarial glycocalyx as well as major immunogen is in progress. against glycoproteins contained in E/S products of circum- and postacetabular glands of cercariae [49]. The glycocalyx of T. regenti cercariae was described as the most antigenic 6. CNS Infection structure and its remnants were still present on 1-day The bird schistosome T. regenti exhibits an unusual mode of old schistosomula transformed in vitro [50], therefore IgM behavior. After successful escape from the host skin, schis- antibodies could recognize components of the glycocalyx tosomula migrate further to the CNS of both specific avian during primary infection. and accidental mammalian hosts [5, 7]. CNS represents an After penetration into the host skin, cercariae transform obligatory part on the migration pathway of T. regenti to the to schistosomula by shedding their tails, releasing E/S pro- nasal mucosa [4]. In specific bird hosts, schistosomula grow ducts, and rebuilding their surface [51]. The dominant and mature during the migration, and their development is part of the cercarial surface is represented by glycocalyx, completed in the nasal area of the host [4]. In accidental which is likely the main component responsible for com- mammalian hosts, the parasite is not able to complete its plement activation [52]. Cercarial surface of human and development and dies as an immature schistosomula in the bird schistosomes is recognized by antibodies from humans spinal cord or brain [4]. and mice infected with T. regenti, T. szidati,andS. mansoni Soon after penetration of cercariae into the skin of [53]. In the skin, parasites need to avoid destruction by specific as well as accidental definitive hosts, schistosomula the host immune system, thus the transformation of cercaria locate peripheral nerves, enter nerve fascicles (Figure 1)or to schistosomulum is accompanied by the decrease of surface epineurium, and reach the spinal cord via spinal roots [5, 57]. saccharides and antigen epitopes which could be recognized Schistosomula appear in the spinal cord from day 2 post by lectins and antibodies, respectively [28]. Although a infection (p.i.); intact parasites can be detected in the duck substantial part of glycocalyx is removed by the cercariae spinal cord even 23 days p.i. [7], and 21–24 days p.i. in the during penetration [54], some of glycocalyx components spinal cord of mice [5]. Then, schistosomula migrate to the remain associated with surface of the schistosomula for some brain where the parasites occur from day 12 p.i. to day 18 p.i. time after transformation [54, 55]. Therefore, not only the in the case of infected ducks, and from 3 days p.i. to 24 days cercarial surface but also the surface of the early in vitro p.i. in the case of infected mice [5]. At the beginning of the transformed (5 h) schistosomula was strongly recognized by brain infection, schistosomula are located mainly in medulla IgG and total Ig antibodies from mice repeatedly infected oblongata and further in subarachnoidal area of cerebellum by T. szidati [28]. E/S products of human as well as [5, 57]. Other localization in the brain is mostly restricted to bird schistosome cercariae, mainly the products released by the subarachnoidal space and the area of the fourth ventricle transforming larvae, are rich in components of glycocalyx [57, 58]. and secretions of circum- and post-acetabular glands [23, Migration through the host CNS requires parasites 54]. adaptations to this environment. Light-brown-pigmented Immunohistochemical staining of ultrathin sections of granules in the intestine of T. regenti schistosomula [59], particular developmental stages revealed variable distribu- which exhibit immunoreactivity with antibodies against tion of antigens recognized by IgG antibodies from sera of components of CNS [57], proved that schistosomula utilize mice re-infected with T. regenti [50]. Except for antibody host nervous tissue for nutrition during their migration via binding to the surface of cercariae and 1-day-old schisto- CNS. For this purpose the parasite should have proteases somula, a positive reaction with spherical bodies located in capable of cleaving components of the nervous tissue. Until subtegumental cells of cercariae and early schistosomula was the present only cathepsins B1 and B2 with the capability Journal of Parasitology Research 5

(a) (b)

Figure 1: T. regenti schistosomula (arrows) migrating inside the peripheral nerve fascicles of an experimentally infected mouse: (a) haematoxylin and eosin staining, scale bar 100 μm; (b) myelin sheets visualized by binding of anti-MBP (myelin basic protein) antibody and secondary anti-rabbit IgG-Cy3 (red), neuroﬁlaments stained by anti-SMI-32 (neuroﬁlament H nonphosphorylated) and secondary anti-mouse IgG-Alexa Fluor (green), cell nuclei stained by DAPI (4,6-diamidino-2-phenylindole) (blue).

to degrade myelin basic protein [26, 27] were identified as CNS [57]. Similarly, microglia are the most important cells candidate molecules. in prevention against Toxoplasma gondii tachyzoite pro- Except localization in CNS that is typical for this species, liferation in the brain [60]. The presence of T. regenti schistosomula were accidentally observed in the lungs of schistosomula in the nervous tissue triggered activation and specific avian [4]aswellasnon-compatiblemammalian proliferation of astrocytes, which were detected in the tracks hosts [5, 57]. Moreover, schistosomula were also found in after migrating schistosomula [57]. Formation of glial scars skin capillaries [53]. However, in vitro tests of blood vessel by astrocytic processes around inflammatory infiltrates was attractiveness did not show any positive results [5]. There- also observed in the brain of human patients infected fore, it seems that schistosomulum presence in the lungs with Taenia solium metacestodes [61]. The presence of the represents an ectopic localization of the parasite in heavily metacestodes was accompanied by astrocytic activation and infected experimental animals [57]. increased expression of glial fibrillary acidic protein (GFAP) [61]. Similar activation of astrocytes was detected in the 7. Immune Response and Pathology in the CNS brain of Toxocara-infected mice [62]. Progress of T. regenti infection led to the formation of inflammatory lesions sur- Presence of the parasites in CNS initiates a cellular response. rounding, destroyed schistosomula in the white matter of the In the spinal cord of ducks 23 days p.i., the parasites were spinal cord. Lesions were formed by microglia, macrophages, located mostly in meninges of thoracic and synsacral spinal eosinophils, neutrophils and CD3+ lymphocytes, and dam- cord and in the white and gray matter of synsacral spinal cord aged axons were detected in the tissue surrounding the [7]. The surroundings of the worms were infiltrated with lesions. On the other hand, presence of the parasites eosinophils, heterophils, and less frequently with plasma outside of parenchyma, in subarachnoidal space of the spinal cells, histiocytes, lymphocytes, and macrophages [7]. Despite cord and brain, and in the cavity of the 4th ventricle of dense infiltration with the immune cells, the parasites were the brain, did not initiate any heavy inflammation, nor not destroyed by host immune response [7]. The para- pathological changes in the surrounding tissue. This implies sites located in brain, predominantly in meninges, were that schistosomula are able to prolong their survival while surrounded by macrophages and endothelial cells [7]. At they migrate outside of parenchyma [57]. Nevertheless, present, details about the immune response in the infected progression of the infection inevitably led to elimination ducks are still missing. of the parasite. Most schistosomula were destroyed on day Morestudieshavebeendoneonmiceasamodelof 21 p.i. [57]. Challenge infections initiated development of non-compatible host. Early infections (3 days p.i.) of the a strong immune response leading to rapid destruction and mouse spinal cord did not initiate any inflammatory reaction elimination of schistosomula. No intact worms, only parasite or damage to the nervous tissue; only focal oedema was remnants surrounded by inflammatory foci, were observed observed [6, 57]. Signs of the infection were observed 6 or 7 in CNS 3 days after the fourth infection [6, 57]. days p.i., when the vicinity of schistosomula was infiltrated Infections of immunodeficient mice (SCID strain) with granulocytes, predominantly neutrophils, activated revealed that deficiency in T- and B-cell production led to microglia, macrophages and, less frequently, CD3+ lympho- development of mild immune reaction, which is not suffi- cytes [6, 57]. It seems that microglia and macrophages, are cient for elimination of the parasite [6, 57]. Even challenge responsible for destruction of schistosomula in the mouse infections did not induce proper immune reactions leading 6 Journal of Parasitology Research to the destruction of all worms. However, the tissue in actively search for their snail host and a new life cycle of T. the vicinity of intact schistosomula was infiltrated with regenti begin. inflammatory cells, and also axonal damage as a result of schistosomulum migration was observed [57]. A higher 9. Conclusion number of schistosomula in CNS and prolonged survival of Avian schistosomes, in comparison to the extensive studies worms indicate the importance of lymphocytes in the on human schistosomes, are neglected. The most studied destruction of schistosomula [57]. Lymphocyte infiltration species of avian schistosomes belong to the genus Tri- in CNS was also demonstrated for other disorders, including chobilharzia. Their life cycle is connected to an aquatic neurocysticercosis [63], cerebral malaria [64], and toxo- environment and they use waterfowl as definitive hosts. plasmic encephalitis [65]. It was observed that T cells Adult worms of the genus Trichobilharzia inhabit either potentiated an immune defense against Mesocestoides corti visceral or nasal areas of their bird hosts. Visceral species infection of mice by indirect activation of other immune cells represent the majority of avian schistosomes, whereas nasal (i.e., macrophages) and resident CNS cells (i.e., microglia, species form a small group. Trichobilharzia regenti is the only astrocytes) [63]. nasal species whose life cycle is described. After penetration The increased number of T. regenti schistosomula mig- into the skin, T. regenti is able to invade peripheral nerves rating through the nervous tissue of immunodeficient mice and continue via CNS to the nasal cavity of birds where adult caused axonal damage and activation and proliferation of females lay eggs. Such neuroinfections of birds can result astrocytes. No significant differences in the number of dam- in transient or permanent neuromotor disorders, especially aged axons around the inflammatory lesions and in the vicin- during heavy infections. ity of schistosomula were observed in immunocompetent Experimental studies on mice revealed that bird schis- and immunodeficient mice. Therefore, axonal injury was tosomes possess an ability to penetrate into the body of probably caused mechanically by migrating schistosomula mammals. In case of nonsensitized or immunodeficient and not by the host immune defense [57]. These injuries of mice, the parasites are not eliminated in the skin by the host the spinal cord resulted in partial hind leg paralysis [57]or immune system and can migrate further through the host even death (unpublished) of infected SCID mice. body; the parasites never mature in mammalian host and die after a few days/weeks after infection. In case of infections with T. regenti, schistosomula reach the spinal cord and 8. Terminal Phase of the Infection brain of the experimental mice. Migrating schistosomula The exact migratory route from the brain to the site of final cause axonal damage in the mouse nervous tissue and initiate location in the nasals is not described. Two hypotheses have development of inflammatory reaction. Neuroinfections, been published. Based on the presence of worms in bulbus mainly in case of immunodeficient animals, are manifest by olfactorius [5, 7] a hypothesis about migration via cranial hind leg paralysis and even death of the heavily infected host. nerves was formulated. Nevertheless, investigation of cranial Under natural conditions, cercariae of bird schistosomes nerves, n. olfactorius and n. opticus, did not reveal any par- are attracted not only by the duck-foot skin, but they asites or lesions; therefore this hypothesis was rejected [58]. also positively react to human skin stimuli. In the human Histological studies showed an extravascular localization of skin, cercariae cause an allergic disease known as cercarial the parasite in subarachnoidal space; several worms were also dermatitis. It develops after repeated contact with cercariae located intravascularly [7, 58]. The intestine of the worms as a response of the host immune system to antigens in meninges contained a dark-brown pigment, probably of penetrating parasites, and leads to the destruction of hematinproducedbyhemoglobindigestion[58, 59]. These parasites in the skin. findings support the theory that T. regenti probably migrates Based on recent reports of bird schistosomes and out- from the meninges to the nasal cavity via blood vessels [58], breaks of cercarial dermatitis in new areas, cercarial dermati- but this hypothesis needs to be validated. tis is considered as a reemerging disease [1]. Experimental infections of small mammals show that there might be a The first appearance of parasites in the nasal mucosa potential health risk for humans due to exposure to T. regenti was noted 13 days p.i. [4]. Intact live worms were located cercariae (particularly for immunodeficient patients). If the intravascularly or extravascularly [66] in the connective tis- parasite is not killed in the skin, then its neurotropic behavior sue between cartilage of turbinate and glandular epithelium might represent a serious problem. Unfortunately, no data of the nasal mucosa until 24 days p.i. [58].Thetissuearound on such human infections are available, and an appropriate the adult worms was infiltrated with various inflammatory diagnostic tool is still missing. Immunological or molecular cells [7]. Fifteen days p.i., immature eggs were detected methods for differential diagnosis of bird schistosomes are extravascularly in connective tissue close to cartilage. The highly desirable for verification/exclusion of T. regenti as a highest number of eggs appeared 22 days p.i. and they were causative agent of undetermined neurological disorders of distributed all over the nasal mucosa [58]. The eggs with fully animal and human patients. developed miracidia were surrounded by a focal dense mass of eosinophils, heterophils, histiocytes, and multinucleated Acknowledgments giant cells [7]. Miracidia hatch directly in the nasal tissue and leave the nasal cavity (bill) when the duck submerges its The authors wish to thank Dr Sara V. Brant (The University bill in water to feed or drink [34]. Swimming miracidia then of New Mexico) for the linguistic corrections. The research Journal of Parasitology Research 7 of coauthors has been supported by the Czech Science Foun- conditions,” Parasitology Research, vol. 107, no. 4, pp. 923–930, dation (Grant no. 502/11/1621) and the Charles University 2010. in Prague (the Projects PRVOUK P41 and UNCE 204017). [16] J. Rudolfova,´ J. Sitko, and P. Horak,´ “Nasal schistosomes of wildfowl in the Czech Republic,” Parasitology Research, vol. 88, no. 12, pp. 1093–1095, 2002. References [17] W. Feiler and W. Haas, “Host-finding in Trichobilharzia ocellata cercariae: swimming and attachment to the host,” Para- [1] P. Horak´ and L. Kola´rovˇ a,´ “Snails, waterfowl and cercarial sitology, vol. 96, no. 3, pp. 493–505, 1988. dermatitis,” Freshwater Biology, vol. 56, no. 4, pp. 779–790, [18] W. Feiler and W. Haas, “Trichobilharzia ocellata: chemical 2011. stimuli of duck skin for cercarial attachment,” Parasitology, [2] P. Kourilovˇ a,´ K. G. Hogg, L. Kola´rovˇ a,´ and A. P. Mountford, vol. 96, no. 3, pp. 507–517, 1988. “Cercarial dermatitis caused by bird schistosomes comprises [19] W. Haas and A. Van De Roemer, “Invasion of the vertebrate both immediate and late phase cutaneous hypersensitivity skin by cercariae of Trichobilharzia ocellata:penetrationpro- reactions,” Journal of Immunology, vol. 172, no. 6, pp. 3766– cesses and stimulating host signals,” Parasitology Research, vol. 3774, 2004. 84, no. 10, pp. 787–795, 1998. [3] P. Horak´ and L. E. Kola´rovˇ a,´ “Bird schistosomes: do they die [20] W. Haas and S. Haeberlein, “Penetration of cercariae into the in mammalian skin?” Trends in Parasitology,vol.17,no.2,pp. living human skin: Schistosoma mansoni vs. Trichobilharzia 66–69, 2001. szidati,” Parasitology Research, vol. 105, no. 4, pp. 1061–1066, [4] P. Horak,J.Dvo´ rˇak,L.Kol´ a´rovˇ a,´ and L. Trefil, “Trichobilharzia 2009. regenti, a pathogen of the avian and mammalian central ner- [21] A. Ligasova,´ J. Bulantova,´ O. Sebesta,ˇ M. Kasnˇ y,´ K. Koberna, vous systems,” Parasitology, vol. 119, no. 6, pp. 577–581, 1999. and L. Mikes,ˇ “Secretory glands in cercaria of the neuropatho- [5] K. Hradkov´ a´ and P. Horak,´ “Neurotropic behaviour of Tri- genic schistosome Trichobilharzia regenti—ultrastructural chobilharzia regenti in ducks and mice,” Journal of Helminthol- characterization, 3-D modelling, volume and pH estimations,” ogy, vol. 76, no. 2, pp. 137–141, 2002. Parasites and Vectors, vol. 4, no. 1, article 162, 2011. [6] P. Kourilovˇ a,´ M. Syru˚cek,ˇ and L. Kola´rovˇ a,´ “The severity of [22]J.P.Salter,K.C.Lim,E.Hansell,I.Hsieh,andJ.H.McKerrow, mouse pathologies caused by the bird schistosome Trichobil- “Schistosome invasion of human skin and degradation of harzia regenti in relation to host immune status,” Parasitology dermal elastin are mediated by a single serine protease,” Research, vol. 93, no. 1, pp. 8–16, 2004. Journal of Biological Chemistry, vol. 275, no. 49, pp. 38667– [7] L. Kola´rovˇ a,´ P. Horak,´ and F. Cada,ˇ “Histopathology of 38673, 2000. CNS and nasal infections caused by Trichobilharzia regenti in [23] L. Mikes,ˇ L. Z`ıdkova,´ M. Kasnˇ y,´ J. Dvorˇak,´ and P. Horak,´ “In vertebrates,” Parasitology Research, vol. 87, no. 8, pp. 644–650, vitro stimulation of penetration gland emptying by Trichobil- 2001. harzia szidati and T. regenti (Schistosomatidae) cercariae. [8]S.V.BrantandE.S.Loker,“Molecularsystematicsofthe Quantitative collection and partial characterization of the pro- avian schistosome genus Trichobilharzia (Trematoda: Schisto- ducts,” Parasitology Research, vol. 96, no. 4, pp. 230–241, 2005. somatidae) in North America,” Journal of Parasitology, vol. 95, [24] M. Kasnˇ y,´ L. Mikes,J.P.Dalton,A.P.Mountford,andˇ no. 4, pp. 941–963, 2009. P. Hor ak,´ “Comparison of cysteine peptidase activities in [9] D. Jouet, H. Fert, C. Hologne, M. L. Kaltenbach, and J. Trichobilharzia regenti and Schistosoma mansoni cercariae,” Depaquit, “Avian schistosomes in French aquatic birds: a Parasitology, vol. 134, no. 11, pp. 1599–1609, 2007. molecular approach,” Journal of Helminthology, vol. 83, no. 2, [25] J. Dvorˇak,´ S. T. Mashiyama, S. Braschi et al., “Differential use pp. 181–189, 2009. of protease families for invasion by schistosome cercariae,” [10] A. Korsunenko, G. Chrisanfova, A. Lopatkin et al., “Genetic Biochimie, vol. 90, no. 2, pp. 345–358, 2008. differentiation of cercariae infrapopulations of the avian [26] J. Dvorˇak,´ M. Delcroix, A. Rossi et al., “Multiple cathepsin schistosome Trichobilharzia szidati based on RAPD markers B isoforms in schistosomula of Trichobilharzia regenti:iden- and mitochondrial cox1 gene,” Parasitology Research, vol. 110, tification, characterisation and putative role in migration and no. 2, pp. 833–841, 2012. nutrition,” International Journal for Parasitology, vol. 35, no. 8, [11] A. V. Korsunenko, G. G. Chrisanfova, A. P. Ryskov, S. O. pp. 895–910, 2005. Movsessian,V.A.Vasilyev,andS.K.Semyenova,“Detection [27] K. Doleckovˇ a,´ M. Kasnˇ y,´ L. Mikesˇ et al., “The functional of European Trichobilharzia schistosomes (T. franki, T. szidati, expression and characterisation of a cysteine peptidase from and T. regenti) based on novel genome sequences,” Journal of the invasive stage of the neuropathogenic schistosome Tri- Parasitology, vol. 96, no. 4, pp. 802–806, 2010. chobilharzia regenti,” International Journal for Parasitology, vol. [12] A. Faltynkov´ a´ and W. Haas, “Larval trematodes in freshwater 39, no. 2, pp. 201–211, 2009. molluscs from the Elbe to Danube rivers (Southeast Ger- [28] P. Horak,´ L. Kova´r,ˇ L. Kola´rovˇ a,´ and J. Nebesa´rovˇ a,´ “Cercaria- many): before and today,” Parasitology Research, vol. 99, no. schistosomulum surface transformation of Trichobilharzia 5, pp. 572–582, 2006. szidati and its putative immunological impact,” Parasitology, [13] H. Ferte,´ J. Depaquit, S. Carre,´ I. Villena, and N. Leger,´ vol. 116, no. 2, pp. 139–147, 1998. “Presence of Trichobilharzia szidati in Lymnaea stagnalis and T. [29] K. Grabe and W. Haas, “Navigation within host tissues: cer- franki in Radix auricularia in northeastern France: molecular cariae orientate towards dark after penetration,” Parasitology evidence,” Parasitology Research, vol. 95, no. 2, pp. 150–154, Research, vol. 93, no. 2, pp. 111–113, 2004. 2005. [30] K. Grabe and W. Haas, “Navigation within host tissues: [14] J. Rudolfova,´ D. T. J. Littlewood, J. Sitko, and P. Horak,´ “Bird Schistosoma mansoni and Trichobilharzia ocellata schistoso- schistosomes of wildfowl in the Czech Republic and Poland,” mula respond to chemical gradients,” International Journal for Folia Parasitologica, vol. 54, no. 2, pp. 88–93, 2007. Parasitology, vol. 34, no. 8, pp. 927–934, 2004. [15] D. Jouet, K. Sk´ırnisson, L. Kola´rovˇ a,´ and H. Ferte,´ “Final [31] L. M. Verbrugge, J. J. Rainey, R. L. Reimink, and H. D. hosts and variability of Trichobilharzia regenti under natural Blankespoor, “Swimmer’s itch: incidence and risk factors,” 8 Journal of Parasitology Research

American Journal of Public Health, vol. 94, no. 5, pp. 738–741, [48] H. Turner and J. P. Kinet, “Signalling through the high-affinity 2004. IgE receptor FcεRI,” Nature, vol. 402, no. 6760, supplement, [32] E. Chamot, L. Toscani, and A. Rougemont, “Public health pp. B24–B30, 1999. importance and risk factors for cercarial dermatitis associated [49] L. Lichtenbergova,´ P. Kolbekova,´ P. Kourilovˇ a´ et al., “Antibody with swimming in Lake Leman at Geneva, Switzerland,” Epide- responses induced by Trichobilharzia regenti antigens in miology and Infection, vol. 120, no. 3, pp. 305–314, 1998. murine and human hosts exhibiting cercarial dermatitis,” [33] D. L. Augustine and T. H. Weller, “Experimental studies on the Parasite Immunology, vol. 30, no. 11-12, pp. 585–595, 2008. specificity of skin tests for the diagnosis of schistosomiasis,” [50] M. Chanova,´ L. Lichtenbergova,´ J. Bulantova,´ L. Mikes,ˇ and P. The Journal of Parasitology, vol. 35, no. 5, pp. 461–466, 1949. Horak,´ “Trichobilharzia regenti: antigenic structures of intra- [34] P. Horak,´ L. Kola´rovˇ a,´ and C. M. Adema, “Biology of the vertebrate stages,” Central European Journal of Biology, vol. 7, schistosome genus Trichobilharzia,” Advances in Parasitology, no. 1, pp. 83–90, 2012. vol. 52, pp. 155–233, 2002. [51] J. C. Samuelson and J. P. Caulfield, “The cercarial glycocalyx [35] L. Kola´rovˇ a,´ V. Gottwaldova,´ D. Cechovˇ a,´ and M. Sevcovˇ a,´ of Schistosoma mansoni,” Journal of Cell Biology, vol. 100, no. “The occurrence of cercarial dermatitis in Central Bohemia,” 5, pp. 1423–1434, 1985. Zentralblatt fur Hygiene und Umweltmedizin, vol. 189, no. 1, [52] J. C. Samuelson and J. P. Caulfield, “Cercarial glycocalyx of pp. 1–13, 1989. Schistosoma mansoni activates human complement,” Infection [36] T. L. Meinking, C. N. Burkhart, and C. G. Burkhart, “Chang- and Immunity, vol. 51, no. 1, pp. 181–186, 1986. ing paradigms in parasitic infections: common dermatological [53] P. Kourilovˇ a´ and L. Kola´rovˇ a,´ “Variations in immunofluores- helminthic infections and cutaneous myiasis,” Clinics in cent antibody response against Trichobilharzia and Schisto- Dermatology, vol. 21, no. 5, pp. 407–416, 2003. soma antigens in compatible and incompatible hosts,” Para- [37] S. Bechtold, U. Wintergerst, and O. Butenandt, “Cercarian sitology Research, vol. 88, no. 6, pp. 513–521, 2002. dermatitis,” Monatsschrift fur Kinderheilkunde, vol. 145, no. 11, [54] J. C. Samuelson and J. P. Caulfield, “Loss of covalently pp. 1170–1172, 1997. labeled glycoproteins and glycolipids from the surface of newly [38] K. Ramaswamy, Y. X. He, and B. Salafsky, “ICAM-1 and iNOS transformed schistosomula of Schistosoma mansoni,” Journal expression increased in the skin of mice after vaccination with of Cell Biology, vol. 94, no. 2, pp. 363–369, 1982. γ-irradiated cercariae of Schistosoma mansoni,” Experimental [55] L. Wang, Y. L. Li, Z. Fishelson, J. R. Kusel, and A. Ruppel, Parasitology, vol. 86, no. 2, pp. 118–132, 1997. “Schistosoma japonicum migration through mouse skin com- [39] M. Bahgat, K. Francklow, M. J. Doenhoff et al., “Infection pared histologically and immunologically with S. mansoni,” induces antibodies against the cercarial secretions, but not Parasitology Research, vol. 95, no. 3, pp. 218–223, 2005. against the cercarial elastases of Schistosoma mansoni, Schis- [56] R. A. Wilson and P. E. Barnes, “The tegument of Schistosoma tosoma haematobium, Schistosoma japonicum and Trichobil- mansoni: observations on the formation, structure and com- harzia ocellata,” Parasite Immunology, vol. 23, no. 10, pp. 557– position of cytoplasmic inclusions in relation to tegument 565, 2001. function,” Parasitology, vol. 68, no. 2, pp. 239–258, 1974. [40] K. G. Hogg, S. Kumkate, and A. P. Mountford, “IL-10 [57] L. Lichtenbergova,´ H. Lassmann, M. K. Jones, L. Kola´rovˇ a,´ regulates early IL-12-mediated immune responses induced by and P. Horak,´ “Trichobilharzia regenti: host immune response the radiation-attenuated schistosome vaccine,” International in the pathogenesis of neuroinfection in mice,” Experimental Immunology, vol. 15, no. 12, pp. 1451–1459, 2003. Parasitology, vol. 128, no. 4, pp. 328–335, 2011. [41] E. V. Acosta-Rodriguez, G. Napolitani, A. Lanzavecchia, and F. [58] M. Chanova´ and P. Horak,´ “Terminal phase of bird schistoso- Sallusto, “Interleukins 1β and 6 but not transforming growth miasis caused by Trichobilharzia regenti (Schistosomatidae) in factor-β are essential for the differentiation of interleukin 17- ducks (Anas platyrhynchos f. domestica),” Folia Parasitologica, producing human T helper cells,” Nature Immunology, vol. 8, vol. 54, no. 2, pp. 105–107, 2007. no. 9, pp. 942–949, 2007. [59] K. Blazovˇ a´ and P. Horak,´ “Trichobilharzia regenti: the develop- [42] F. H. Falcone, H. Haas, and B. F. Gibbs, “The human basophil: mental differences in natural and abnormal hosts,” Parasitol- a new appreciation of its role in immune responses,” Blood, ogy International, vol. 54, no. 3, pp. 167–172, 2005. vol. 96, no. 13, pp. 4028–4038, 2000. [60] R. B. Rock, G. Gekker, S. Hu et al., “Role of microglia in central [43]T.KawakamiandS.J.Galli,“Regulationofmast-celland nervous system infections,” Clinical Microbiology Reviews, vol. basophil function and survival by IgE,” Nature Reviews Immu- 17, no. 4, pp. 942–964, 2004. nology, vol. 2, no. 10, pp. 773–786, 2002. [61] J. I. Alvarez, C. H. Colegial, C. A. Castao, J. Trujillo, J. M. Teale, [44] Y. Banu and T. Watanabe, “Augmentation of antigen receptor- and B. I. Restrepo, “The human nervous tissue in proximity to mediated responses by histamine H1 receptor signaling,” granulomatous lesions induced by Taenia solium metacestodes Journal of Experimental Medicine, vol. 189, no. 4, pp. 673–682, displays an active response,” Journal of Neuroimmunology, vol. 1999. 127, no. 1-2, pp. 139–144, 2002. [45] M. Jutel, T. Watanabe, S. Klunker et al., “Histamine regulates [62]A.A.Othman,G.A.Abdel-Aleem,E.M.Saied,W.W.Mayah, T-cell and antibody responses by differential expression of H1 and A. M. Elatrash, “Biochemical and immunopathological and H2 receptors,” Nature, vol. 413, no. 6854, pp. 420–425, changes in experimental neurotoxocariasis,” Molecular and 2001. Biochemical Parasitology, vol. 172, no. 1, pp. 1–8, 2010. [46] M. Rincon,´ J. Anguita, T. Nakamura, E. Fikrig, and R. A. [63] A. E. Cardona and J. M. Teale, “γ/δ T cell-deficient mice Flavell, “Interleukin (IL)-6 directs the differentiation of IL- exhibit reduced disease severity and decreased inflammatory 4-producing CD4+ Tcells,”Journal of Experimental Medicine, response in the brain in murine neurocysticercosis,” Journal of vol. 185, no. 3, pp. 461–469, 1997. Immunology, vol. 169, no. 6, pp. 3163–3171, 2002. [47] F. H. Falcone, C. A. Dahinden, B. F. Gibbs et al., “Human [64] D. S. Hansen, N. J. Bernard, C. Q. Nie, and L. Scholeld, basophils release interleukin-4 after stimulation with Schisto- “NK cells stimulate recruitment of CXCR3+ T cells to the soma mansoni egg antigen,” European Journal of Immunology, brain during Plasmodium berghei-mediated cerebral malaria,” vol. 26, no. 5, pp. 1147–1155, 1996. Journal of Immunology, vol. 178, no. 9, pp. 5779–5788, 2007. Journal of Parasitology Research 9

[65] E. H. Wilson, U. Wille-Reece, F. Dzierszinski, and C. A. Hunter, “A critical role for IL-10 in limiting inﬂammation during toxoplasmic encephalitis,” Journal of Neuroimmunology, vol. 165, no. 1-2, pp. 63–74, 2005. [66] P. Horak,L.Kol´ a´rovˇ a,´ and J. Dvorˇak,´ “Trichobilharzia regenti n. sp. (Schistosomatidae, Bilharziellinae), a new nasal schistosome from Europe,” Parasite, vol. 5, no. 4, pp. 349–357, 1998.